LOCUS       sequence-162432-       10340 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses huAsCpf1-P2A-puro and crRNA guide.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10340)
  AUTHORS   Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, 
            DeGennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS, 
            Wu WY, Scott DA, Severinov K, van der Oost J, Zhang F
  TITLE     Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array.
  JOURNAL   Nat Biotechnol. 2017 Jan;35(1):31-34. doi: 10.1038/nbt.3737. Epub 
            2016 Dec 5.
  PUBMED    27918548
REFERENCE   2  (bases 1 to 10340)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt.3737"; 
            journalName: "Nat Biotechnol"; date: "2017-01"; volume: "35"; issue:
            "1"; pages: "31-34"
COMMENT     Sequence Label: pY30
FEATURES             Location/Qualifiers
     source          1..10340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        170..418
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     170..190
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     341..360
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        538..917
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        918..1121
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1072..1092
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1166..1185
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1166..1184
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1232..5152
                     /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
                     VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQ
                     FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYD
                     VKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIEN
                     HRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQM
                     RNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKES
                     KDLKLQNGISNQDWLAYIQELRN"
     regulatory      4872..4881
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5153..5200
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5210..5266
                     /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             5267..5863
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     5760..5780
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5934..5951)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5940..6164
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6210..6638
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6297..6316)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6507..6528
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6647..6667)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6652..6981
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6832..6967
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6894..6913
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             7048..7842
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7102..7121)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7712..7731
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    8016..8137
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8053..8072)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8107..8126
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8186..8202)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8186..8202)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8199..8221)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8210..8226
                     /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)."
     promoter        complement(join(8234..8240,8241..8258,8259..8264))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8279..8300
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8417..8434)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8588..9173)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8668..8687)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9344..10135,10136..10204))
                     /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     9967..9986
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10205..10309)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga cttaattaag agggcctatt
      181 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
      241 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
      301 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
      361 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgt
      421 aatttctact cttgtagatg gagacgatat atcgtctctt tttcaattgc atgaagaatc
      481 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      541 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      601 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      661 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      721 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      781 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      841 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      901 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      961 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     1021 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     1081 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac
     1141 ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct
     1201 agcgtttaaa cttaagcttg gtaccgccac catgacacag ttcgagggct ttaccaacct
     1261 gtatcaggtg agcaagacac tgcggtttga gctgatccca cagggcaaga ccctgaagca
     1321 catccaggag cagggcttca tcgaggagga caaggcccgc aatgatcact acaaggagct
     1381 gaagcccatc atcgatcgga tctacaagac ctatgccgac cagtgcctgc agctggtgca
     1441 gctggattgg gagaacctga gcgccgccat cgactcctat agaaaggaga aaaccgagga
     1501 gacaaggaac gccctgatcg aggagcaggc cacatatcgc aatgccatcc acgactactt
     1561 catcggccgg acagacaacc tgaccgatgc catcaataag agacacgccg agatctacaa
     1621 gggcctgttc aaggccgagc tgtttaatgg caaggtgctg aagcagctgg gcaccgtgac
     1681 cacaaccgag cacgagaacg ccctgctgcg gagcttcgac aagtttacaa cctacttctc
     1741 cggcttttat gagaacagga agaacgtgtt cagcgccgag gatatcagca cagccatccc
     1801 acaccgcatc gtgcaggaca acttccccaa gtttaaggag aattgtcaca tcttcacacg
     1861 cctgatcacc gccgtgccca gcctgcggga gcactttgag aacgtgaaga aggccatcgg
     1921 catcttcgtg agcacctcca tcgaggaggt gttttccttc cctttttata accagctgct
     1981 gacacagacc cagatcgacc tgtataacca gctgctggga ggaatctctc gggaggcagg
     2041 caccgagaag atcaagggcc tgaacgaggt gctgaatctg gccatccaga agaatgatga
     2101 gacagcccac atcatcgcct ccctgccaca cagattcatc cccctgttta agcagatcct
     2161 gtccgatagg aacaccctgt ctttcatcct ggaggagttt aagagcgacg aggaagtgat
     2221 ccagtccttc tgcaagtaca agacactgct gagaaacgag aacgtgctgg agacagccga
     2281 ggccctgttt aacgagctga acagcatcga cctgacacac atcttcatca gccacaagaa
     2341 gctggagaca atcagcagcg ccctgtgcga ccactgggat acactgagga atgccctgta
     2401 tgagcggaga atctccgagc tgacaggcaa gatcaccaag tctgccaagg agaaggtgca
     2461 gcgcagcctg aagcacgagg atatcaacct gcaggagatc atctctgccg caggcaagga
     2521 gctgagcgag gccttcaagc agaaaaccag cgagatcctg tcccacgcac acgccgccct
     2581 ggatcagcca ctgcctacaa ccctgaagaa gcaggaggag aaggagatcc tgaagtctca
     2641 gctggacagc ctgctgggcc tgtaccacct gctggactgg tttgccgtgg atgagtccaa
     2701 cgaggtggac cccgagttct ctgcccggct gaccggcatc aagctggaga tggagccttc
     2761 tctgagcttc tacaacaagg ccagaaatta tgccaccaag aagccctact ccgtggagaa
     2821 gttcaagctg aactttcaga tgcctacact ggcctctggc tgggacgtga ataaggagaa
     2881 gaacaatggc gccatcctgt ttgtgaagaa cggcctgtac tatctgggca tcatgccaaa
     2941 gcagaagggc aggtataagg ccctgagctt cgagcccaca gagaaaacca gcgagggctt
     3001 tgataagatg tactatgact acttccctga tgccgccaag atgatcccaa agtgcagcac
     3061 ccagctgaag gccgtgacag cccactttca gacccacaca acccccatcc tgctgtccaa
     3121 caatttcatc gagcctctgg agatcacaaa ggagatctac gacctgaaca atcctgagaa
     3181 ggagccaaag aagtttcaga cagcctacgc caagaaaacc ggcgaccaga agggctacag
     3241 agaggccctg tgcaagtgga tcgacttcac aagggatttt ctgtccaagt ataccaagac
     3301 aacctctatc gatctgtcta gcctgcggcc atcctctcag tataaggacc tgggcgagta
     3361 ctatgccgag ctgaatcccc tgctgtacca catcagcttc cagagaatcg ccgagaagga
     3421 gatcatggat gccgtggaga caggcaagct gtacctgttc cagatctata acaaggactt
     3481 tgccaagggc caccacggca agcctaatct gcacacactg tattggaccg gcctgttttc
     3541 tccagagaac ctggccaaga caagcatcaa gctgaatggc caggccgagc tgttctaccg
     3601 ccctaagtcc aggatgaaga ggatggcaca ccggctggga gagaagatgc tgaacaagaa
     3661 gctgaaggat cagaaaaccc caatccccga caccctgtac caggagctgt acgactatgt
     3721 gaatcacaga ctgtcccacg acctgtctga tgaggccagg gccctgctgc ccaacgtgat
     3781 caccaaggag gtgtctcacg agatcatcaa ggataggcgc tttaccagcg acaagttctt
     3841 tttccacgtg cctatcacac tgaactatca ggccgccaat tccccatcta agttcaacca
     3901 gagggtgaat gcctacctga aggagcaccc cgagacacct atcatcggca tcgatcgggg
     3961 cgagagaaac ctgatctata tcacagtgat cgactccacc ggcaagatcc tggagcagcg
     4021 gagcctgaac accatccagc agtttgatta ccagaagaag ctggacaaca gggagaagga
     4081 gagggtggca gcaaggcagg cctggtctgt ggtgggcaca atcaaggatc tgaagcaggg
     4141 ctatctgagc caggtcatcc acgagatcgt ggacctgatg atccactacc aggccgtggt
     4201 ggtgctggag aacctgaatt tcggctttaa gagcaagagg accggcatcg ccgagaaggc
     4261 cgtgtaccag cagttcgaga agatgctgat cgataagctg aattgcctgg tgctgaagga
     4321 ctatccagca gagaaagtgg gaggcgtgct gaacccatac cagctgacag accagttcac
     4381 ctcctttgcc aagatgggca cccagtctgg cttcctgttt tacgtgcctg ccccatatac
     4441 atctaagatc gatcccctga ccggcttcgt ggaccccttc gtgtggaaaa ccatcaagaa
     4501 tcacgagagc cgcaagcact tcctggaggg cttcgacttt ctgcactacg acgtgaaaac
     4561 cggcgacttc atcctgcact ttaagatgaa cagaaatctg tccttccaga ggggcctgcc
     4621 cggctttatg cctgcatggg atatcgtgtt cgagaagaac gagacacagt ttgacgccaa
     4681 gggcacccct ttcatcgccg gcaagagaat cgtgccagtg atcgagaatc acagattcac
     4741 cggcagatac cgggacctgt atcctgccaa cgagctgatc gccctgctgg aggagaaggg
     4801 catcgtgttc agggatggct ccaacatcct gccaaagctg ctggagaatg acgattctca
     4861 cgccatcgac accatggtgg ccctgatccg cagcgtgctg cagatgcgga actccaatgc
     4921 cgccacaggc gaggactata tcaacagccc cgtgcgcgat ctgaatggcg tgtgcttcga
     4981 ctcccggttt cagaacccag agtggcccat ggacgccgat gccaatggcg cctaccacat
     5041 cgccctgaag ggccagctgc tgctgaatca cctgaaggag agcaaggatc tgaagctgca
     5101 gaacggcatc tccaatcagg actggctggc ctacatccag gagctgcgca acaaaaggcc
     5161 ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag ggatccggcg caacaaactt
     5221 ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgaccg agtacaagcc
     5281 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     5341 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     5401 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     5461 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     5521 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     5581 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     5641 caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     5701 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     5761 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     5821 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc taagaattct gcagatatcc
     5881 agcacagtgg cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     5941 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     6001 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     6061 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     6121 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     6181 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     6241 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     6301 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     6361 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     6421 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     6481 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     6541 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     6601 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     6661 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     6721 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     6781 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     6841 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     6901 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     6961 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     7021 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     7081 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     7141 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     7201 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     7261 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     7321 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     7381 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     7441 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     7501 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     7561 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     7621 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     7681 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     7741 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     7801 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     7861 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     7921 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     7981 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     8041 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     8101 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     8161 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     8221 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     8281 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     8341 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     8401 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     8461 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     8521 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     8581 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     8641 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     8701 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     8761 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     8821 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     8881 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     8941 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     9001 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     9061 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     9121 gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     9181 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     9241 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     9301 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     9361 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     9421 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     9481 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     9541 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     9601 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     9661 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     9721 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     9781 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     9841 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     9901 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     9961 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
    10021 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
    10081 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
    10141 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
    10201 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
    10261 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
    10321 gaaaagtgcc acctgacgtc
//
