LOCUS       sequence-426482-       11797 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac expression of dAsCas12a-3xKRAB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11797)
  AUTHORS   Hsiung CC, Wilson CM, Sambold NA, Dai R, Chen Q, Teyssier N, 
            Misiukiewicz S, Arab A, O'Loughlin T, Cofsky JC, Shi J, Gilbert LA
  TITLE     Engineered CRISPR-Cas12a for higher-order combinatorial chromatin 
            perturbations.
  JOURNAL   Nat Biotechnol. 2024 May 17. doi: 10.1038/s41587-024-02224-0.
  PUBMED    38760567
REFERENCE   2  (bases 1 to 11797)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2024 May 17. doi: 10.1038/s41587-024-02224-0."
COMMENT     Sequence Label: pCH68 (dAsCas12a-3xKRAB piggyBac)
FEATURES             Location/Qualifiers
     source          1..11797
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        147..526
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        528..804
                     /label=chicken beta-actin promoter
     intron          805..1821
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1745..1767
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1829..1848
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1887..5807
                     /codon_start=1
                     /product="CRISPR-associated protein from Acidaminococcus 
                     sp. BV3L6 (Zetsche et al., 2015)"
                     /label=AsCpf1
                     /translation="MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND
                     HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRN
                     AIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFD
                     KFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHF
                     ENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL
                     NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL
                     RNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG
                     KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL
                     KKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA
                     RNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYK
                     ALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEP
                     LEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSID
                     LSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKG
                     HHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLK
                     DQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFF
                     HVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR
                     SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV
                     VVLANLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQ
                     FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYD
                     VKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIEN
                     HRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQM
                     RNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKES
                     KDLKLQNGISNQDWLAYIQELRN"
     regulatory      5527..5536
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5808..5855
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5868..6053
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             6066..6251
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             6264..6449
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             6456..6536
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             6546..6602
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6603..7310
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     misc_feature    7350..7938
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7403..7423)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7821..7832)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7977..7994)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7983..8207
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8561..8595
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(8682..8701)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8683..8701)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8708..8725)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8708..8724)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8717..8739)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(8866..9321)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9003..9024)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     9215..9234
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        9348..9452
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9453..9521,9522..10313)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(9671..9690)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10484..11072
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10973..10992
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11226..11243
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11360..11381
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11396..11401,11402..11419,11420..11426)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11434..11450
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     11439..11461
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11458..11474
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11458..11474
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     11493..11513
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        11495..11513
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   11577..11639
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 aagtaacaaa acttttaaac attctctctt ttacaaaaat aaacttattt tgtactttaa
       61 aaacagtcat gttgtattat aaaataagta attagcttaa cctatacata atagaaacaa
      121 attatactta ttaaccggtc atggtcgaca ttgattattg actagttatt aatagtaatc
      181 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      241 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      301 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      361 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      421 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      481 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      541 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      601 tattttttaa ttattttgtg cagcgatggg ggcggggggg ggggggggcg cgcgccaggc
      661 ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat
      721 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat
      781 aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc
      841 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg
      901 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt
      961 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg
     1021 gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc
     1081 gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt
     1141 gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg
     1201 aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg
     1261 gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg
     1321 ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg
     1381 caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg
     1441 cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt
     1501 ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg
     1561 aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc
     1621 ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc
     1681 cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg
     1741 gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat
     1801 gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat
     1861 tttggcaaag aattgctagc gccaccatga cacagttcga gggctttacc aacctgtatc
     1921 aggtgagcaa gacactgcgg tttgagctga tcccacaggg caagaccctg aagcacatcc
     1981 aggagcaggg cttcatcgag gaggacaagg cccgcaatga tcactacaag gagctgaagc
     2041 ccatcatcga tcggatctac aagacctatg ccgaccagtg cctgcagctg gtgcagctgg
     2101 attgggagaa cctgagcgcc gccatcgact cctatagaaa ggagaaaacc gaggagacaa
     2161 ggaacgccct gatcgaggag caggccacat atcgcaatgc catccacgac tacttcatcg
     2221 gccggacaga caacctgacc gatgccatca ataagagaca cgccgagatc tacaagggcc
     2281 tgttcaaggc cgagctgttt aatggcaagg tgctgaagca gctgggcacc gtgaccacaa
     2341 ccgagcacga gaacgccctg ctgcggagct tcgacaagtt tacaacctac ttctccggct
     2401 tttatgagaa caggaagaac gtgttcagcg ccgaggatat cagcacagcc atcccacacc
     2461 gcatcgtgca ggacaacttc cccaagttta aggagaattg tcacatcttc acacgcctga
     2521 tcaccgccgt gcccagcctg cgggagcact ttgagaacgt gaagaaggcc atcggcatct
     2581 tcgtgagcac ctccatcgag gaggtgtttt ccttcccttt ttataaccag ctgctgacac
     2641 agacccagat cgacctgtat aaccagctgc tgggaggaat ctctcgggag gcaggcaccg
     2701 agaagatcaa gggcctgaac gaggtgctga atctggccat ccagaagaat gatgagacag
     2761 cccacatcat cgcctccctg ccacacagat tcatccccct gtttaagcag atcctgtccg
     2821 ataggaacac cctgtctttc atcctggagg agtttaagag cgacgaggaa gtgatccagt
     2881 ccttctgcaa gtacaagaca ctgctgagaa acgagaacgt gctggagaca gccgaggccc
     2941 tgtttaacga gctgaacagc atcgacctga cacacatctt catcagccac aagaagctgg
     3001 agacaatcag cagcgccctg tgcgaccact gggatacact gaggaatgcc ctgtatgagc
     3061 ggagaatctc cgagctgaca ggcaagatca ccaagtctgc caaggagaag gtgcagcgca
     3121 gcctgaagca cgaggatatc aacctgcagg agatcatctc tgccgcaggc aaggagctga
     3181 gcgaggcctt caagcagaaa accagcgaga tcctgtccca cgcacacgcc gccctggatc
     3241 agccactgcc tacaaccctg aagaagcagg aggagaagga gatcctgaag tctcagctgg
     3301 acagcctgct gggcctgtac cacctgctgg actggtttgc cgtggatgag tccaacgagg
     3361 tggaccccga gttctctgcc cggctgaccg gcatcaagct ggagatggag ccttctctga
     3421 gcttctacaa caaggccaga aattatgcca ccaagaagcc ctactccgtg gagaagttca
     3481 agctgaactt tcagatgcct acactggcct ctggctggga cgtgaataag gagaagaaca
     3541 atggcgccat cctgtttgtg aagaacggcc tgtactatct gggcatcatg ccaaagcaga
     3601 agggcaggta taaggccctg agcttcgagc ccacagagaa aaccagcgag ggctttgata
     3661 agatgtacta tgactacttc cctgatgccg ccaagatgat cccaaagtgc agcacccagc
     3721 tgaaggccgt gacagcccac tttcagaccc acacaacccc catcctgctg tccaacaatt
     3781 tcatcgagcc tctggagatc acaaaggaga tctacgacct gaacaatcct gagaaggagc
     3841 caaagaagtt tcagacagcc tacgccaaga aaaccggcga ccagaagggc tacagagagg
     3901 ccctgtgcaa gtggatcgac ttcacaaggg attttctgtc caagtatacc aagacaacct
     3961 ctatcgatct gtctagcctg cggccatcct ctcagtataa ggacctgggc gagtactatg
     4021 ccgagctgaa tcccctgctg taccacatca gcttccagag aatcgccgag aaggagatca
     4081 tggatgccgt ggagacaggc aagctgtacc tgttccagat ctataacaag gactttgcca
     4141 agggccacca cggcaagcct aatctgcaca cactgtattg gaccggcctg ttttctccag
     4201 agaacctggc caagacaagc atcaagctga atggccaggc cgagctgttc taccgcccta
     4261 agtccaggat gaagaggatg gcacaccggc tgggagagaa gatgctgaac aagaagctga
     4321 aggatcagaa aaccccaatc cccgacaccc tgtaccagga gctgtacgac tatgtgaatc
     4381 acagactgtc ccacgacctg tctgatgagg ccagggccct gctgcccaac gtgatcacca
     4441 aggaggtgtc tcacgagatc atcaaggata ggcgctttac cagcgacaag ttctttttcc
     4501 acgtgcctat cacactgaac tatcaggccg ccaattcccc atctaagttc aaccagaggg
     4561 tgaatgccta cctgaaggag caccccgaga cacctatcat cggcatcgat cggggcgaga
     4621 gaaacctgat ctatatcaca gtgatcgact ccaccggcaa gatcctggag cagcggagcc
     4681 tgaacaccat ccagcagttt gattaccaga agaagctgga caacagggag aaggagaggg
     4741 tggcagcaag gcaggcctgg tctgtggtgg gcacaatcaa ggatctgaag cagggctatc
     4801 tgagccaggt catccacgag atcgtggacc tgatgatcca ctaccaggcc gtggtggtgc
     4861 tggccaacct gaatttcggc tttaagagca agaggaccgg catcgccgag aaggccgtgt
     4921 accagcagtt cgagaagatg ctgatcgata agctgaattg cctggtgctg aaggactatc
     4981 cagcagagaa agtgggaggc gtgctgaacc cataccagct gacagaccag ttcacctcct
     5041 ttgccaagat gggcacccag tctggcttcc tgttttacgt gcctgcccca tatacatcta
     5101 agatcgatcc cctgaccggc ttcgtggacc ccttcgtgtg gaaaaccatc aagaatcacg
     5161 agagccgcaa gcacttcctg gagggcttcg actttctgca ctacgacgtg aaaaccggcg
     5221 acttcatcct gcactttaag atgaacagaa atctgtcctt ccagaggggc ctgcccggct
     5281 ttatgcctgc atgggatatc gtgttcgaga agaacgagac acagtttgac gccaagggca
     5341 cccctttcat cgccggcaag agaatcgtgc cagtgatcga gaatcacaga ttcaccggca
     5401 gataccggga cctgtatcct gccaacgagc tgatcgccct gctggaggag aagggcatcg
     5461 tgttcaggga tggctccaac atcctgccaa agctgctgga gaatgacgat tctcacgcca
     5521 tcgacaccat ggtggccctg atccgcagcg tgctgcagat gcggaactcc aatgccgcca
     5581 caggcgagga ctatatcaac agccccgtgc gcgatctgaa tggcgtgtgc ttcgactccc
     5641 ggtttcagaa cccagagtgg cccatggacg ccgatgccaa tggcgcctac cacatcgccc
     5701 tgaagggcca gctgctgctg aatcacctga aggagagcaa ggatctgaag ctgcagaacg
     5761 gcatctccaa tcaggactgg ctggcctaca tccaggagct gcgcaacaaa aggccggcgg
     5821 ccacgaaaaa ggccggccag gcaaaaaaga aaaagggatc cggtgggcgg acccttgtta
     5881 ctttcaagga tgtattcgtg gatttcacac gcgaggagtg gaaattgttg gatactgccc
     5941 agcagattgt ataccgcaat gtaatgctcg aaaactacaa aaatctcgtg agtctcggat
     6001 atcaactgac caagcctgac gttatcctcc gattggagaa aggcgaggaa cctgggagcg
     6061 gaggaaggac gcttgtcacg tttaaggatg tttttgttga cttcactcga gaggagtgga
     6121 agctgttgga tactgcccaa caaattgtgt atcgcaacgt gatgctcgag aactacaaga
     6181 accttgtgtc tttgggctat cagctgacta aacctgatgt aatactccgc ctcgagaaag
     6241 gagaggagcc tggtagtggg ggacggaccc tggtaacatt caaggatgta ttcgtggatt
     6301 tcacgcggga agaatggaaa ctcctcgata ctgcccaaca gatagtgtat cgaaacgtaa
     6361 tgctcgaaaa ctacaaaaac ttggtcagcc tcggatacca acttacaaaa cctgatgtta
     6421 tcctcagact tgagaagggc gaggaacccg gatcctaccc atacgatgtt ccagattacg
     6481 cttatcccta cgacgtgcct gattatgcat acccatatga tgtccccgac tatgccagcg
     6541 gccgcgctac taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac
     6601 ctgtgtctaa gggcgaagag ctgattaagg agaacatgca catgaagctg tacatggagg
     6661 gcaccgtgga caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg
     6721 gcacccagac catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca
     6781 tcctggctac tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc
     6841 ccgacttctt caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg
     6901 aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct
     6961 acaacgtcaa gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa
     7021 cactcggctg ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca
     7081 gaaacgacat ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac gccaagacca
     7141 catatagatc caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact
     7201 acagactgga aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg
     7261 cagtggccag atactgcgac ctccctagca aactggggca caagcttaat taattaatta
     7321 atgatcatac agtgctctga gtacgctata atcaacctct ggattacaaa atttgtgaaa
     7381 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     7441 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     7501 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     7561 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     7621 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     7681 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     7741 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     7801 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     7861 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     7921 tttgggccgc ctccccgcag gatccactag agcggccgcc tagagctcgc tatcagcctc
     7981 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     8041 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     8101 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     8161 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     8221 aagaaccagc tggggctcga ctagagcttg cggaaccctt aagctttttt attatatatt
     8281 agtcacgata tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat
     8341 gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat
     8401 gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac
     8461 aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga
     8521 ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag
     8581 actatctttc tagggttaaa aaagatttgc gctttactcg acctaaactt taaacacgtc
     8641 atagaatctt cgtttgacaa aaaccacatt gtgttgggtc gccctatagt gagtcgtatt
     8701 acaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     8761 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     8821 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcaaatt gtaagcgtta
     8881 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     8941 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     9001 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     9061 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     9121 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     9181 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     9241 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     9301 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     9361 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     9421 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     9481 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     9541 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     9601 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     9661 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     9721 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     9781 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     9841 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     9901 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     9961 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
    10021 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
    10081 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
    10141 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
    10201 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
    10261 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
    10321 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
    10381 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
    10441 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
    10501 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
    10561 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
    10621 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
    10681 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
    10741 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
    10801 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
    10861 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
    10921 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
    10981 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
    11041 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
    11101 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
    11161 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
    11221 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
    11281 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
    11341 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
    11401 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
    11461 gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacctc
    11521 ccccggtatt cacgacagca ggctgaataa taaaaaaatt agaaactatt atttaaccct
    11581 agaaagataa tcatattgtg acgtacgtta aagataatca tgcgtaaaat tgacgcatgt
    11641 gttttatcgg tctgtatatc gaggtttatt tattaatttg aatagatatt aagttttatt
    11701 atatttacac ttacatacta ataataaatt caacaaacaa tttatttatg tttatttatt
    11761 tattaaaaaa aaacaaaaac tcaaaatttc ttctata
//
