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"role": "user",
"content": "You are given a reasoning chain that explains and justifies a particular conclusion or answer. Your task is to extract **all distinct knowledge pieces** from this chain. A knowledge piece is any standalone statement conveying an explicit fact, definition, mechanism, relationship, or insight that can be generalized beyond the specific question.\n\n## Instructions:\n1. Read the entire reasoning chain.\n2. Identify each discrete fact or insight expressed.\n3. Rewrite each as a self-contained, generalizable sentence.\n4. Do **not** include any contextual or example-specific details.\n5. Output **only** a list of those sentences.\n\n## Output Format:\n1. knowledge-Piece-1\n2. knowledge-Piece-2\n...\n\n## Reasoning Chain:\nOkay, let's try to figure out which primer is best for Sanger sequencing across the gRNA TU. Hmm, the question mentions Golden Gate cloning with BsmBI, and the goal is to create a transcriptional unit (TU) for a CRISPR guide RNA. So, first, I need to understand how Golden Gate cloning works here. Golden Gate typically involves overlapping oligonucleotides that can anneal and be cut by BsmBI to assemble the DNA. The plasmid has a sequence provided, and the two oligos are being cloned into it.\n\nThe key here is that Sanger sequencing needs to span the gRNA TU. So, the primer should anneal to a region that spans the junction of the two oligos in the plasmid. Since it's a TU for CRISPR gRNA, the sequence would likely be part of the guide RNA sequence. The two oligos are being annealed and cloned, so their combined sequences should form the gRNA TU. Therefore, the primer should be designed to hybridize to a region that's present in both the original plasmid and the gRNA TU, but not in either of the oligos alone. Alternatively, it should target the overlap between the two oligos after they're inserted into the plasmid.\n\nLooking at the options, let's analyze each one. The answer choices are A, B, C, D with their sequences. Let me write them down:\n\nA) TGGTGAAAGTTGGAACCTCTTA\nB) TAAGAGGTTCCAACTTTCACCA\nC) GCTGGCCTTTTGCTCACATG\nD) CATGTGAGCAAAAGGCCAGC\n\nFirst, I need to check the plasmid sequence and see where the oligos would insert. The plasmid sequence is quite long, but maybe I can find the relevant part. The Golden Gate cloning would require the two oligos to overlap so that when they are annealed and cut by BsmBI, their overlapping regions are removed, leaving the desired TU. The BsmBI sites in the oligos would need to be flanked by the overhangs from the plasmid.\n\nWait, in Golden Gate cloning, each assembly reaction uses two restriction enzymes, usually BsaI or BsmBI, which recognize overhangs of 4-6 bases. The idea is that each fragment has a overhang at one end (from one restriction site) and a complementary overhang at the other end (from another restriction site). When you mix them, they can anneal and the internal restriction sites are removed by the enzyme, leaving the desired ligation.\n\nSo, the plasmid pLAB050 has a BsmBI site where the TU is being inserted. The two oligos must have sequences that complement the plasmid's overhang and each other's overhangs. The oligos provided are GACTTTCATCACTATCTTTTCCCCCG and AAACCGGGGGAAAAGATAGTGATGAA. Let's look at their sequences.\n\nFirst, let's check the 5' and 3' ends of the oligos. The first oligo is 5'-GACTTTCATCACTATCTTTTCCCCCG-3'. The second is 5'-AAACCGGGGGAAAAGATAGTGATGAA-3'. The BsmBI recognition sequence is GCGGCCG, which is 6 bases. So, each overhang should be 4-6 bases. Let's see the ends of the oligos.\n\nThe first oligo ends with TTTTTTCCCCCG (assuming it's 5' to 3'). Wait, actually, the first oligo is given as GACTTTCATCACTATCTTTTCCCCCG. Let me confirm the direction. Typically, the sequence is written 5' to 3', so the first nucleotide is G, last is G. But when they are annealed and inserted into the plasmid, their 5' ends would be adjacent to the plasmid's overhang.\n\nAlternatively, maybe the plasmid has a BsmBI site, and the oligos are designed to replace a portion of the plasmid sequence. But the question is about Sanger sequencing across the TU. So, the primer should anneal to a region that is part of the original plasmid and part of the inserted oligos, allowing to sequence through the junction.\n\nLooking at the plasmid sequence, there's a section: ...GGCCTTTTGCTCACATGTT... (Wait, let me check the plasmid sequence given. The user provided a very long plasmid sequence. Let me see if I can find the BsmBI site. BsmBI cuts at GCGGCCG. Let me scan the plasmid sequence for that sequence.\n\nLooking at the plasmid sequence: The user wrote:\n\nTCG...GCGGCCG...TTCAGT... (I'm just making up the middle part) but actually, the user provided the entire sequence. Wait, but that's a lot. Maybe it's easier to look for the BsmBI site. The user mentions that the plasmid was used with Golden Gate cloning, so it must have compatible overhangs. The original plasmid probably has a BsmBI site, and the oligos have overhangs complementary to that.\n\nAlternatively, maybe the plasmid has a specific sequence at the cloning site where the oligos are inserted. The Golden Gate reaction would involve cutting the plasmid with BsmBI and adding overhangs, then annealing the oligos which also have BsmBI overhangs, replacing the original fragment.\n\nBut perhaps the goal is to design a primer that can anneal across the junction. So, if the two oligos are inserted into the plasmid, their combined sequences form the TU. The primer needs to target a region that is part of the plasmid and part of the inserted oligos. So, the primer would bind to a sequence that is present in both the original plasmid and the inserted DNA.\n\nAlternatively, the TU is the inserted part, so the primer should be inside the TU. But since the TU is created by inserting the two oligos, the primer should be designed to span the junction between the plasmid and the inserted oligos.\n\nLooking at the options, let's check each sequence against the plasmid's sequence. Option C is GCTGGCCTTTTGCTCACATG. Let me search the plasmid sequence for this. Wait, the plasmid sequence given is very long. Let me see if I can find \"GCTGGCCTTTTGCTCACATG\" within it.\n\nLooking through the plasmid sequence: The user wrote the sequence as:\n\nTCGGTCTCC... [rest of the sequence] ...GCTGGCCTTTTGCTCACATGTT... [rest] ...GCTTCC... Hmm, actually, this is tedious. Alternatively, maybe the correct primer is one that is part of the original plasmid and part of the inserted oligos, so that when you sequence across the TU, the primer would bind to the plasmid's sequence and then the inserted oligos.\n\nAlternatively, maybe the TU is the combined sequence of the two oligos. The first oligo is 5'-GACTTTCATCACTATCTTTTCCCCCG-3', and the second is 5'-AAACCGGGGGAAAAGATAGTGATGAA-3'. So when they are annealed, their combined TU would be:\n\nGACTTTCATCACTATCTTTTCCCCCGAAACCGGGGGAAAAGATAGTGATGAA\n\nSo the total length of the TU is the sum of both oligos minus the overlapping region. Wait, but the sequences are given as 21 bases each (assuming standard oligo lengths). Let me check the sequences:\n\nFirst oligo: GACTTTCATCACTATCTTTTCCCCC (21 nt)\nSecond oligo: AAACCGGGGGAAAAGATAGTGATGAA (21 nt)\n\nBut the overlapping region would be where they are annealed. The first oligo ends with CCCCC, and the second starts with AAAC. So the overlap is maybe 3-4 bases? Let's see:\n\nFirst oligo 3' end: CCCCC\nSecond oligo 5' end: AAAC\n\nSo the overlapping region would be CCCCC -> AAAC? Wait, but DNA is 5' to 3', so the 3' end of the first oligo is CCCCC, and the 5' end of the second is AAAC. The overlapping bases would be the last few bases of the first oligo and the first few bases of the second. So perhaps the first few bases of the second oligo (AAAC) are complementary to the last few bases of the first. But in reality, the exact overlap depends on the sequences. Let's check. The first oligo's 3' end is CCCCC. The second oligo's 5' end is AAAC. So the 3' end of the first is CCCCC, and the 5' end of the second is AAAC. These are not complementary. So maybe the overlap is somewhere else. Wait, maybe the first oligo has a different 3' end. Let me check again.\n\nWait, the first oligo is written as GACTTTCATCACTATCTTTTCCCCCG. Let's count the nucleotides. G(1), A(2), C(3), T(4), T(5), T(6), C(7), A(8), C(9), T(10), A(11), T(12), T(13), T(14), T(15), T(16), T(17), C(18), C(19), C(20), G(21). So the 3' end is G. The second oligo is AAACCGGGGGAAAAGATAGTGATGAA. 5' end is A, then A, A, C. So 5' end of second is AAAC. The 3' end of first is G. Not complementary. Therefore, the overlap must be in the middle.\n\nAlternatively, maybe the first oligo is designed to have a 5' overhang and a 3' overhang that matches the plasmid's BsmBI site. Similarly, the second oligo would have a 5' overhang that matches the other BsmBI site. So when they are inserted into the plasmid, the overlapping region between the two oligos would be excised by BsmBI, leaving the TU.\n\nBut for Sanger sequencing, the primer needs to span the junction. So the primer should be designed to bind a sequence that is present in the original plasmid and in the TU. Alternatively, the primer could bind to a region within the TU but designed to start at the plasmid insertion site.\n\nLooking at the options, let's see:\n\nOption C: GCTGGCCTTTTGCTCACATG. Let's check if this sequence is present in both the plasmid and the TU. If the TU is the two oligos inserted into the plasmid, then the TU would have the sequences of both oligos. So the TU's DNA would be:\n\nFirst oligo: GACTTTCATCACTATCTTTTCCCCCG\nSecond oligo: AAACCGGGGGAAAAGATAGTGATGAA\n\nCombined, the TU would be:\n\nGACTTTCATCACTATCTTTTCCCCCGAAACCGGGGGAAAAGATAGTGATGAA\n\nNow, looking for the presence of a primer in this combined sequence. The options are the four primers. Let's check each.\n\nOption A: TGGTGAAAGTTGGAACCTCTTA. Let's see if this sequence is present in the TU. The TU is 21 + 21 = 42 nt. Let's check if A's sequence is part of that.\n\nLooking at the TU sequence:\n\n5' G A C T T T C A T C A C T A T C D T T T C C C G G A A A C C G G G G G A A A G A A A G A T A G T G A T G A A G G C T T C G G T T A T T A A T T C A T C G G A A A T T C C G G T T C C T G G C C C A A T C C G G A A A C T G G G G G C C T G C C T T G C C T G C C T C T T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C C T G C...\n\n## Now perform the extraction."
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{
"text": "I need to extract all distinct knowledge pieces from this reasoning chain. Let me carefully read through the chain and identify all the standalone facts, definitions, mechanisms, relationships, or insights that can be generalized beyond the specific question.\n\nFirst, let me understand what the question is about: It seems to be about Sanger sequencing across a gRNA TU (transcriptional unit) for Golden Gate cloning with BsmBI. The person is trying to figure out which primer is best for Sanger sequencing across the gRNA TU.\n\nLet me go through the text and extract all the distinct knowledge pieces:\n\n1. Golden Gate cloning involves overlapping oligonucleotides that can anneal and be cut by BsmBI to assemble DNA.\n2. The plasmid has a sequence where the TU (transcriptional unit) is being inserted.\n3. Sanger sequencing needs to span the gRNA TU.\n4. The primer should anneal to a region that spans the junction of the two oligos in the plasmid.\n5. The primer should target a sequence present in both the original plasmid and the inserted oligos, but not in either oligo alone.\n6. Alternatively, it should target the overlap between the two oligos after they're inserted into the plasmid.\n7. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG (6 bases).\n8. Golden Gate cloning typically uses restriction enzymes with overhangs of 4-6 bases.\n9. Each fragment (oligo) has a 5' overhang that matches the plasmid's overhang and a complementary overhang for the other fragment.\n10. The plasmid sequence contains a BsmBI site where the TU is being inserted.\n11. The two oligos (first oligo: GACTTTCATCACTATCTTTTCCCCCG and second oligo: AAACCGGGGGAAAAGATAGTGATGAA) are designed to insert into the plasmid.\n12. The first oligo ends with CCCCC (3' end), and the second oligo starts with AAAC (5' end).\n13. The combined TU would be the sequence of the first oligo followed by the second oligo (with some overlap).\n14. The primer should be designed to bind to a sequence that is part of the original plasmid and part of the inserted oligos.\n\nLet me try to rephrase these as self-contained, generalizable sentences without any context or example-specific details:\n\n1. Golden Gate cloning utilizes overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.\n2. Sanger sequencing requires primers that span the junction of the transcriptional unit (TU) in the target DNA.\n3. An optimal primer for Sanger sequencing across a gRNA TU should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.\n4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.\n5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.\n6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.\n7. In Golden Gate cloning, each fragment has a 5' overhang that is complementary to the overhang of another fragment.\n8. The sequence of the inserted DNA fragment in Golden Gate cloning consists of two oligonucleotides joined at an overlap region.\n\nWait, I need to be careful to extract only the knowledge pieces that are explicitly stated in the reasoning chain and can be generalized.\n\nLet me go through the text again more carefully:\n\nFrom the chain:\n- \"Golden Gate typically involves overlapping oligonucleotides that can anneal and be cut by BsmBI to assemble the DNA.\"\n- \"The plasmid has a sequence provided, and the two oligos are being cloned into it.\"\n- \"Sanger sequencing needs to span the gRNA TU.\"\n- \"The primer should anneal to a region that spans the junction of the two oligos in the plasmid.\"\n- \"Since it's a TU for CRISPR gRNA, the sequence would likely be part of the guide RNA sequence.\"\n- \"The two oligos are being annealed and cloned, so their combined sequences should form the gRNA TU.\"\n- \"Therefore, the primer should be designed to hybridize to a region that's present in both the original plasmid and the gRNA TU, but not in either of the oligos alone.\"\n- \"Alternatively, it should target the overlap between the two oligos after they're inserted into the plasmid.\"\n- \"BsmBI recognition sequence is GCGGCCG (6 bases).\"\n- \"In Golden Gate cloning, each assembly reaction uses two restriction enzymes, usually BsaI or BsmBI, which recognize overhangs of 4-6 bases.\"\n- \"The idea is that each fragment has a overhang at one end (from one restriction site) and a complementary overhang at the other end (from another restriction site). When you mix them, they can anneal and the internal restriction sites are removed by the enzyme, leaving the desired ligation.\"\n\nLet me extract the distinct knowledge pieces:\n\n1. Golden Gate cloning involves overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.\n2. Sanger sequencing requires primers that span the junction of the transcriptional unit (TU) in the target DNA.\n3. An optimal primer for Sanger sequencing across a gRNA TU should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.\n4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.\n5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.\n6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.\n7. In Golden Gate cloning, each DNA fragment has a 5' overhang that is complementary to the overhang of another fragment.\n8. In Golden Gate cloning, the internal restriction sites are removed by the enzyme after the fragments anneal, leaving the desired ligation.\n\nWait, I need to be careful about what's explicitly stated. Let me go through the text again more systematically.\n\nThe text says:\n- \"Golden Gate typically involves overlapping oligonucleotides that can anneal and be cut by BsmBI to assemble the DNA.\"\n- \"The primer should anneal to a region that spans the junction of the two oligos in the plasmid.\"\n- \"The two oligos are being annealed and cloned, so their combined sequences should form the gRNA TU.\"\n- \"Therefore, the primer should be designed to hybridize to a region that's present in both the original plasmid and the gRNA TU, but not in either of the oligos alone.\"\n- \"Alternatively, it should target the overlap between the two oligos after they're inserted into the plasmid.\"\n- \"BsmBI recognition sequence is GCGGCCG (6 bases).\"\n- \"In Golden Gate cloning, each assembly reaction uses two restriction enzymes, usually BsaI or BsmBI, which recognize overhangs of 4-6 bases.\"\n- \"The idea is that each fragment has a overhang at one end (from one restriction site) and a complementary overhang at the other end (from another restriction site). When you mix them, they can anneal and the internal restriction sites are removed by the enzyme, leaving the desired ligation.\"\n\nLet me create a list of distinct knowledge pieces:\n\n1. Golden Gate cloning involves overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.\n2. Sanger sequencing requires primers that span the junction of the transcriptional unit (TU) in the target DNA.\n3. An optimal primer for Sanger sequencing across a gRNA TU should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.\n4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.\n5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.\n6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.\n7. In Golden Gate cloning, each DNA fragment has an overhang at one end that is complementary to the overhang of another fragment.\n8. In Golden Gate cloning, the internal restriction sites are removed by the enzyme after the fragments anneal, leaving the desired ligation.\n\nLet me check if I'm missing anything. I think these are the distinct knowledge pieces that can be generalized beyond the specific question.\n\nI should make sure each is a standalone, self-contained sentence that conveys an explicit fact, definition, mechanism, relationship, or insight.\n\nLet me rephrase them to be more precise and general:\n\n1. Golden Gate cloning utilizes overlapping oligonucleotides that anneal and are cleaved by restriction enzymes to assemble DNA fragments.\n2. Sanger sequencing requires primers that span the junction of the transcriptional unit in the target DNA.\n3. An optimal primer for Sanger sequencing across a gRNA transcriptional unit should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.\n4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.\n5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.\n6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.\n7. In Golden Gate cloning, each DNA fragment has a 5' overhang that is complementary to the overhang of another fragment.\n8. In Golden Gate cloning, the internal restriction sites are excised by the enzyme after the fragments anneal, leaving the desired ligation.\n\nI think these are the distinct knowledge pieces. Let me double-check to make sure I'm not including any context or example-specific details.\n\nFor example, the text mentions specific oligo sequences (GACTTTCATCACTATCTTTTCCCCCG and AAACCGGGGGAAAAGATAGTGATGAA), but I shouldn't include those since they're specific to this problem.\n\nAlso, the text mentions that the primer should be designed to hybridize to a region that's present in both the original plasmid and the gRNA TU, but not in either of the oligos alone. I've captured that in point 3.\n\nLet me review once more to ensure I have all the distinct knowledge pieces.\n\n1. Golden Gate cloning involves overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.\n2. Sanger sequencing requires primers that span the junction of the transcriptional unit (TU) in the target DNA.\n3. An optimal primer for Sanger sequencing across a gRNA TU should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.\n4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.\n5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.\n6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.\n7. In Golden Gate cloning, each DNA fragment has an overhang at one end that is complementary to the overhang of another fragment.\n8. In Golden Gate cloning, the internal restriction sites are removed by the enzyme after the fragments anneal, leaving the desired ligation.\n\nI think these 8 points cover all the distinct knowledge pieces from the reasoning chain.\n\nLet me write them as clean, self-contained sentences:\n\n1. Golden Gate cloning involves overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.\n2. Sanger sequencing requires primers that span the junction of the transcriptional unit in the target DNA.\n3. An optimal primer for Sanger sequencing across a gRNA transcriptional unit should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.\n4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.\n5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.\n6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.\n7. In Golden Gate cloning, each DNA fragment has a complementary overhang at one end that pairs with the overhang of another fragment.\n8. In Golden Gate cloning, the internal restriction sites are excised by the enzyme after the fragments anneal, leaving the desired ligation.\n\nI think these are all the distinct knowledge pieces that can be generalized beyond the specific question.\n\nLet me check if there's anything else:\n\n- The text mentions \"BsmBI recognition sequence is GCGGCCG (6 bases)\" - I've captured this in point 5.\n- The text mentions \"each assembly reaction uses two restriction enzymes, usually BsaI or BsmBI\" - I've captured this in point 6 by saying \"typically employs restriction enzymes that generate overhangs of 4-6 base pairs\" (since BsaI and BsmBI both generate 4-6 base overhangs).\n- The text mentions \"each fragment has a overhang at one end (from one restriction site) and a complementary overhang at the other end (from another restriction site)\" - I've captured this in point 7.\n- The text mentions \"when you mix them, they can anneal and the internal restriction sites are removed by the enzyme, leaving the desired ligation\" - I've captured this in point 8.\n\nI think these are all the distinct knowledge pieces.\n\nLet me format the output exactly as requested: just the list of knowledge pieces, each numbered with a hyphen, in the specified format.\n\nThe output should be:\n1. knowledge-piece-1\n2. knowledge-piece-2\n...\n\nSo my list would be:\n1. Golden Gate cloning involves overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.\n2. Sanger sequencing requires primers that span the junction of the transcriptional unit in the target DNA.\n3. An optimal primer for Sanger sequencing across a gRNA transcriptional unit should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.\n4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.\n5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.\n6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.\n7. In Golden Gate cloning, each DNA fragment has a complementary overhang at one end that pairs with the overhang of another fragment.\n8. In Golden Gate cloning, the internal restriction sites are excised by the enzyme after the fragments anneal, leaving the desired ligation.\n\nI need to make sure each is a standalone, self-contained sentence without any context or example-specific details.\n\nLet me review each one:\n\n1. This is a general statement about Golden Gate cloning that can be generalized beyond the specific question.\n2. This is a general statement about Sanger sequencing requirements.\n3. This is a general statement about optimal primer design for Sanger sequencing across a gRNA TU.\n4. This is a general statement about where the optimal primer should target.\n5. This is a specific fact about BsmBI's recognition sequence.\n6. This is a general statement about Golden Gate cloning's overhangs.\n7. This is a general statement about the mechanism of Golden Gate cloning.\n8. This is a general statement about the outcome of Golden Gate cloning.\n\nYes, these all seem to be distinct knowledge pieces that can be generalized.\n\nI'll output them in the required format.\n </think> \n\n1. Golden Gate cloning involves overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.\n2. Sanger sequencing requires primers that span the junction of the transcriptional unit in the target DNA.\n3. An optimal primer for Sanger sequencing across a gRNA transcriptional unit should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.\n4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.\n5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.\n6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.\n7. In Golden Gate cloning, each DNA fragment has a complementary overhang at one end that pairs with the overhang of another fragment.\n8. In Golden Gate cloning, the internal restriction sites are excised by the enzyme after the fragments anneal, leaving the desired ligation."
}
|
I need to extract all distinct knowledge pieces from this reasoning chain. Let me carefully read through the chain and identify all the standalone facts, definitions, mechanisms, relationships, or insights that can be generalized beyond the specific question.
First, let me understand what the question is about: It seems to be about Sanger sequencing across a gRNA TU (transcriptional unit) for Golden Gate cloning with BsmBI. The person is trying to figure out which primer is best for Sanger sequencing across the gRNA TU.
Let me go through the text and extract all the distinct knowledge pieces:
1. Golden Gate cloning involves overlapping oligonucleotides that can anneal and be cut by BsmBI to assemble DNA.
2. The plasmid has a sequence where the TU (transcriptional unit) is being inserted.
3. Sanger sequencing needs to span the gRNA TU.
4. The primer should anneal to a region that spans the junction of the two oligos in the plasmid.
5. The primer should target a sequence present in both the original plasmid and the inserted oligos, but not in either oligo alone.
6. Alternatively, it should target the overlap between the two oligos after they're inserted into the plasmid.
7. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG (6 bases).
8. Golden Gate cloning typically uses restriction enzymes with overhangs of 4-6 bases.
9. Each fragment (oligo) has a 5' overhang that matches the plasmid's overhang and a complementary overhang for the other fragment.
10. The plasmid sequence contains a BsmBI site where the TU is being inserted.
11. The two oligos (first oligo: GACTTTCATCACTATCTTTTCCCCCG and second oligo: AAACCGGGGGAAAAGATAGTGATGAA) are designed to insert into the plasmid.
12. The first oligo ends with CCCCC (3' end), and the second oligo starts with AAAC (5' end).
13. The combined TU would be the sequence of the first oligo followed by the second oligo (with some overlap).
14. The primer should be designed to bind to a sequence that is part of the original plasmid and part of the inserted oligos.
Let me try to rephrase these as self-contained, generalizable sentences without any context or example-specific details:
1. Golden Gate cloning utilizes overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.
2. Sanger sequencing requires primers that span the junction of the transcriptional unit (TU) in the target DNA.
3. An optimal primer for Sanger sequencing across a gRNA TU should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.
4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.
5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.
6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.
7. In Golden Gate cloning, each fragment has a 5' overhang that is complementary to the overhang of another fragment.
8. The sequence of the inserted DNA fragment in Golden Gate cloning consists of two oligonucleotides joined at an overlap region.
Wait, I need to be careful to extract only the knowledge pieces that are explicitly stated in the reasoning chain and can be generalized.
Let me go through the text again more carefully:
From the chain:
- "Golden Gate typically involves overlapping oligonucleotides that can anneal and be cut by BsmBI to assemble the DNA."
- "The plasmid has a sequence provided, and the two oligos are being cloned into it."
- "Sanger sequencing needs to span the gRNA TU."
- "The primer should anneal to a region that spans the junction of the two oligos in the plasmid."
- "Since it's a TU for CRISPR gRNA, the sequence would likely be part of the guide RNA sequence."
- "The two oligos are being annealed and cloned, so their combined sequences should form the gRNA TU."
- "Therefore, the primer should be designed to hybridize to a region that's present in both the original plasmid and the gRNA TU, but not in either of the oligos alone."
- "Alternatively, it should target the overlap between the two oligos after they're inserted into the plasmid."
- "BsmBI recognition sequence is GCGGCCG (6 bases)."
- "In Golden Gate cloning, each assembly reaction uses two restriction enzymes, usually BsaI or BsmBI, which recognize overhangs of 4-6 bases."
- "The idea is that each fragment has a overhang at one end (from one restriction site) and a complementary overhang at the other end (from another restriction site). When you mix them, they can anneal and the internal restriction sites are removed by the enzyme, leaving the desired ligation."
Let me extract the distinct knowledge pieces:
1. Golden Gate cloning involves overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.
2. Sanger sequencing requires primers that span the junction of the transcriptional unit (TU) in the target DNA.
3. An optimal primer for Sanger sequencing across a gRNA TU should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.
4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.
5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.
6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.
7. In Golden Gate cloning, each DNA fragment has a 5' overhang that is complementary to the overhang of another fragment.
8. In Golden Gate cloning, the internal restriction sites are removed by the enzyme after the fragments anneal, leaving the desired ligation.
Wait, I need to be careful about what's explicitly stated. Let me go through the text again more systematically.
The text says:
- "Golden Gate typically involves overlapping oligonucleotides that can anneal and be cut by BsmBI to assemble the DNA."
- "The primer should anneal to a region that spans the junction of the two oligos in the plasmid."
- "The two oligos are being annealed and cloned, so their combined sequences should form the gRNA TU."
- "Therefore, the primer should be designed to hybridize to a region that's present in both the original plasmid and the gRNA TU, but not in either of the oligos alone."
- "Alternatively, it should target the overlap between the two oligos after they're inserted into the plasmid."
- "BsmBI recognition sequence is GCGGCCG (6 bases)."
- "In Golden Gate cloning, each assembly reaction uses two restriction enzymes, usually BsaI or BsmBI, which recognize overhangs of 4-6 bases."
- "The idea is that each fragment has a overhang at one end (from one restriction site) and a complementary overhang at the other end (from another restriction site). When you mix them, they can anneal and the internal restriction sites are removed by the enzyme, leaving the desired ligation."
Let me create a list of distinct knowledge pieces:
1. Golden Gate cloning involves overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.
2. Sanger sequencing requires primers that span the junction of the transcriptional unit (TU) in the target DNA.
3. An optimal primer for Sanger sequencing across a gRNA TU should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.
4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.
5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.
6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.
7. In Golden Gate cloning, each DNA fragment has an overhang at one end that is complementary to the overhang of another fragment.
8. In Golden Gate cloning, the internal restriction sites are removed by the enzyme after the fragments anneal, leaving the desired ligation.
Let me check if I'm missing anything. I think these are the distinct knowledge pieces that can be generalized beyond the specific question.
I should make sure each is a standalone, self-contained sentence that conveys an explicit fact, definition, mechanism, relationship, or insight.
Let me rephrase them to be more precise and general:
1. Golden Gate cloning utilizes overlapping oligonucleotides that anneal and are cleaved by restriction enzymes to assemble DNA fragments.
2. Sanger sequencing requires primers that span the junction of the transcriptional unit in the target DNA.
3. An optimal primer for Sanger sequencing across a gRNA transcriptional unit should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.
4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.
5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.
6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.
7. In Golden Gate cloning, each DNA fragment has a 5' overhang that is complementary to the overhang of another fragment.
8. In Golden Gate cloning, the internal restriction sites are excised by the enzyme after the fragments anneal, leaving the desired ligation.
I think these are the distinct knowledge pieces. Let me double-check to make sure I'm not including any context or example-specific details.
For example, the text mentions specific oligo sequences (GACTTTCATCACTATCTTTTCCCCCG and AAACCGGGGGAAAAGATAGTGATGAA), but I shouldn't include those since they're specific to this problem.
Also, the text mentions that the primer should be designed to hybridize to a region that's present in both the original plasmid and the gRNA TU, but not in either of the oligos alone. I've captured that in point 3.
Let me review once more to ensure I have all the distinct knowledge pieces.
1. Golden Gate cloning involves overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.
2. Sanger sequencing requires primers that span the junction of the transcriptional unit (TU) in the target DNA.
3. An optimal primer for Sanger sequencing across a gRNA TU should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.
4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.
5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.
6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.
7. In Golden Gate cloning, each DNA fragment has an overhang at one end that is complementary to the overhang of another fragment.
8. In Golden Gate cloning, the internal restriction sites are removed by the enzyme after the fragments anneal, leaving the desired ligation.
I think these 8 points cover all the distinct knowledge pieces from the reasoning chain.
Let me write them as clean, self-contained sentences:
1. Golden Gate cloning involves overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.
2. Sanger sequencing requires primers that span the junction of the transcriptional unit in the target DNA.
3. An optimal primer for Sanger sequencing across a gRNA transcriptional unit should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.
4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.
5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.
6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.
7. In Golden Gate cloning, each DNA fragment has a complementary overhang at one end that pairs with the overhang of another fragment.
8. In Golden Gate cloning, the internal restriction sites are excised by the enzyme after the fragments anneal, leaving the desired ligation.
I think these are all the distinct knowledge pieces that can be generalized beyond the specific question.
Let me check if there's anything else:
- The text mentions "BsmBI recognition sequence is GCGGCCG (6 bases)" - I've captured this in point 5.
- The text mentions "each assembly reaction uses two restriction enzymes, usually BsaI or BsmBI" - I've captured this in point 6 by saying "typically employs restriction enzymes that generate overhangs of 4-6 base pairs" (since BsaI and BsmBI both generate 4-6 base overhangs).
- The text mentions "each fragment has a overhang at one end (from one restriction site) and a complementary overhang at the other end (from another restriction site)" - I've captured this in point 7.
- The text mentions "when you mix them, they can anneal and the internal restriction sites are removed by the enzyme, leaving the desired ligation" - I've captured this in point 8.
I think these are all the distinct knowledge pieces.
Let me format the output exactly as requested: just the list of knowledge pieces, each numbered with a hyphen, in the specified format.
The output should be:
1. knowledge-piece-1
2. knowledge-piece-2
...
So my list would be:
1. Golden Gate cloning involves overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.
2. Sanger sequencing requires primers that span the junction of the transcriptional unit in the target DNA.
3. An optimal primer for Sanger sequencing across a gRNA transcriptional unit should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.
4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.
5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.
6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.
7. In Golden Gate cloning, each DNA fragment has a complementary overhang at one end that pairs with the overhang of another fragment.
8. In Golden Gate cloning, the internal restriction sites are excised by the enzyme after the fragments anneal, leaving the desired ligation.
I need to make sure each is a standalone, self-contained sentence without any context or example-specific details.
Let me review each one:
1. This is a general statement about Golden Gate cloning that can be generalized beyond the specific question.
2. This is a general statement about Sanger sequencing requirements.
3. This is a general statement about optimal primer design for Sanger sequencing across a gRNA TU.
4. This is a general statement about where the optimal primer should target.
5. This is a specific fact about BsmBI's recognition sequence.
6. This is a general statement about Golden Gate cloning's overhangs.
7. This is a general statement about the mechanism of Golden Gate cloning.
8. This is a general statement about the outcome of Golden Gate cloning.
Yes, these all seem to be distinct knowledge pieces that can be generalized.
I'll output them in the required format.
</think>
1. Golden Gate cloning involves overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.
2. Sanger sequencing requires primers that span the junction of the transcriptional unit in the target DNA.
3. An optimal primer for Sanger sequencing across a gRNA transcriptional unit should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.
4. The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.
5. BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.
6. Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.
7. In Golden Gate cloning, each DNA fragment has a complementary overhang at one end that pairs with the overhang of another fragment.
8. In Golden Gate cloning, the internal restriction sites are excised by the enzyme after the fragments anneal, leaving the desired ligation.
|
[
"Golden Gate cloning involves overlapping oligonucleotides that anneal and are cut by restriction enzymes to assemble DNA fragments.",
"Sanger sequencing requires primers that span the junction of the transcriptional unit in the target DNA.",
"An optimal primer for Sanger sequencing across a gRNA transcriptional unit should anneal to a sequence present in both the original plasmid and the inserted DNA fragment.",
"The optimal primer should target the region of overlap between the two oligonucleotides after they are inserted into the plasmid.",
"BsmBI is a restriction enzyme with a recognition sequence of GCGGCCG.",
"Golden Gate cloning typically employs restriction enzymes that generate overhangs of 4-6 base pairs.",
"In Golden Gate cloning, each DNA fragment has a complementary overhang at one end that pairs with the overhang of another fragment.",
"In Golden Gate cloning, the internal restriction sites are excised by the enzyme after the fragments anneal, leaving the desired ligation."
] | 0
|
[{"role":"user","content":"You are given a reasoning chain that explains and justifies a particular (...TRUNCATED)
| {"text":"I need to extract all distinct knowledge pieces from this reasoning chain. Let me carefully(...TRUNCATED)
| "I need to extract all distinct knowledge pieces from this reasoning chain. Let me carefully read th(...TRUNCATED)
| ["Golden Gate cloning uses Type IIS restriction enzymes to assemble DNA fragments without cutting th(...TRUNCATED)
| 1
|
[{"role":"user","content":"You are given a reasoning chain that explains and justifies a particular (...TRUNCATED)
| {"text":"I need to extract all distinct knowledge pieces from this reasoning chain. Let me carefully(...TRUNCATED)
| "I need to extract all distinct knowledge pieces from this reasoning chain. Let me carefully go thro(...TRUNCATED)
| ["Golden Gate cloning employs type II restriction enzymes that cleave DNA to produce blunt ends.","G(...TRUNCATED)
| 2
|
[{"role":"user","content":"You are given a reasoning chain that explains and justifies a particular (...TRUNCATED)
| {"text":"First, I need to extract all distinct knowledge pieces from the reasoning chain. A knowledg(...TRUNCATED)
| "First, I need to extract all distinct knowledge pieces from the reasoning chain. A knowledge piece (...TRUNCATED)
| ["Golden Gate cloning enables the simultaneous cloning of multiple DNA fragments without the need fo(...TRUNCATED)
| 3
|
[{"role":"user","content":"You are given a reasoning chain that explains and justifies a particular (...TRUNCATED)
| {"text":"I need to extract all distinct knowledge pieces from this reasoning chain. Let me go throug(...TRUNCATED)
| "I need to extract all distinct knowledge pieces from this reasoning chain. Let me go through it car(...TRUNCATED)
| ["Golden Gate cloning utilizes overlapping ends of DNA inserts to facilitate ligation without requir(...TRUNCATED)
| 4
|
[{"role":"user","content":"You are given a reasoning chain that explains and justifies a particular (...TRUNCATED)
| {"text":"I need to extract all distinct knowledge pieces from the given reasoning chain. Let me care(...TRUNCATED)
| "I need to extract all distinct knowledge pieces from the given reasoning chain. Let me carefully re(...TRUNCATED)
| ["Golden Gate cloning typically uses Type IIs restriction enzymes.","Type IIs restriction enzymes ha(...TRUNCATED)
| 5
|
[{"role":"user","content":"You are given a reasoning chain that explains and justifies a particular (...TRUNCATED)
| {"text":"I need to extract all distinct knowledge pieces from this reasoning chain. A knowledge piec(...TRUNCATED)
| "I need to extract all distinct knowledge pieces from this reasoning chain. A knowledge piece is a s(...TRUNCATED)
| ["I need to extract all distinct knowledge pieces from this reasoning chain. A knowledge piece is a (...TRUNCATED)
| 6
|
[{"role":"user","content":"You are given a reasoning chain that explains and justifies a particular (...TRUNCATED)
| {"text":"I need to extract all distinct knowledge pieces from the reasoning chain. Let me go through(...TRUNCATED)
| "I need to extract all distinct knowledge pieces from the reasoning chain. Let me go through the tex(...TRUNCATED)
| ["TAAACC is the 5' end sequence of the T7 promoter.","AAGCTT is the 3' end sequence of the T7 promot(...TRUNCATED)
| 7
|
[{"role":"user","content":"You are given a reasoning chain that explains and justifies a particular (...TRUNCATED)
| {"text":"I need to extract all distinct knowledge pieces from the reasoning chain. Let me carefully (...TRUNCATED)
| "I need to extract all distinct knowledge pieces from the reasoning chain. Let me carefully read thr(...TRUNCATED)
| ["BamHI is a restriction enzyme that recognizes specific DNA sequences and cuts to produce compatibl(...TRUNCATED)
| 8
|
[{"role":"user","content":"You are given a reasoning chain that explains and justifies a particular (...TRUNCATED)
| {"text":"I need to extract all distinct knowledge pieces from this reasoning chain. The knowledge pi(...TRUNCATED)
| "I need to extract all distinct knowledge pieces from this reasoning chain. The knowledge pieces sho(...TRUNCATED)
| ["Proline is encoded by CCU, CCA, or CCG.","Leucine is encoded by CTT, CTA, or CTG.","To correct a p(...TRUNCATED)
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