LOCUS       sequence-490266-       11589 bp    DNA     circular SYN 22-OCT-2025
DEFINITION  Expresses human codon-optimized Cas9 protein and puromycin 
            resistance from EFS promoter and an AAVS1-targeting sgRNA element 
            from U6 promoter. Lentiviral backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11589)
  AUTHORS   Wang T, Birsoy K, Hughes NW, Krupczak KM, Post Y, Wei JJ, Lander ES,
            Sabatini DM
  TITLE     Identification and characterization of essential genes in the human 
            genome.
  JOURNAL   Science. 2015 Oct 15. pii: aac7041.
  PUBMED    26472758
REFERENCE   2  (bases 1 to 11589)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 22, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2015 Oct 15. pii: aac7041."
COMMENT     Sequence Label: lentiCRISPR - AAVS1 sgRNA
FEATURES             Location/Qualifiers
     source          1..11589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1058..1284
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1285..1465
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1512..1637
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2130..2363
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2548..2592
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        2770..3018
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2770..2790
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2941..2960
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3039..3114
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    3162..3279
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3348..3559
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3579..3588
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3588..3611
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3618..3638
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3663..7763
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             3663..7763
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7764..7811
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7827..7883
                     /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             7884..8483
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7884..7903)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     8380..8400
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8499..9087
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8552..8572)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8970..8981)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9159..9392
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    9470..9591
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9507..9526)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9561..9580
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      9631..9766
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9786..9805)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9787..9805)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9815..9832)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9815..9831)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9824..9846)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9973..10428
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10060..10079)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10270..10291
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10454..10558
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10559..10627,10628..11419)
                     /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(10777..10796)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
     1081 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
     1141 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
     1201 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
     1261 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
     1321 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     1381 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1441 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     1501 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1561 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1621 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1681 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1741 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1801 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1861 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1921 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1981 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     2041 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     2101 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     2161 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     2221 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     2281 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     2341 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     2401 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     2461 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     2521 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     2581 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     2641 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     2701 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     2761 ggacccagag agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2821 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
     2881 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     2941 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     3001 tggaaaggac gaaacaccgg ggccactagg gacaggatgt tttagagcta gaaatagcaa
     3061 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     3121 gaattctaga tcttgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg
     3181 attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact
     3241 aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc
     3301 agagatccac tttggcgccg gctcgagtgg ctccggtgcc cgtcagtggg cagagcgcac
     3361 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag
     3421 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     3481 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     3541 gtttgccgcc agaacacagg tgtcgtgacg cgggatccgc caccatggat tacaaagacg
     3601 atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag
     3661 ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga
     3721 tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc
     3781 acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg
     3841 ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct
     3901 atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac
     3961 tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca
     4021 acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga
     4081 aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca
     4141 tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg
     4201 tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca
     4261 tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac
     4321 ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc
     4381 tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg
     4441 atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc
     4501 agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc
     4561 tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta
     4621 tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc
     4681 agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg
     4741 gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg
     4801 aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga
     4861 agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg
     4921 ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg
     4981 agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca
     5041 gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag
     5101 tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga
     5161 acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt
     5221 ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga
     5281 gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg
     5341 tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct
     5401 ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta
     5461 tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc
     5521 tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc
     5581 acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca
     5641 ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg
     5701 atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca
     5761 gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc
     5821 acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag
     5881 tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga
     5941 tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa
     6001 tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg
     6061 tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg
     6121 atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata
     6181 tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg
     6241 acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga
     6301 actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga
     6361 ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc
     6421 tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca
     6481 ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca
     6541 agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact
     6601 accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt
     6661 accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga
     6721 tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca
     6781 acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc
     6841 ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg
     6901 ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc
     6961 agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg
     7021 ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct
     7081 attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga
     7141 aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact
     7201 ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt
     7261 actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc
     7321 agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc
     7381 actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac
     7441 agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga
     7501 tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc
     7561 ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc
     7621 ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag
     7681 aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg
     7741 acctgtctca gctgggaggc gacaagcgtc ctgctgctac taagaaagct ggtcaagcta
     7801 agaaaaagaa agctagcggc agcggcgcca ccaacttcag cctgctgaag caggccggcg
     7861 acgtggagga gaaccccggc cccatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     7921 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     7981 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     8041 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     8101 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     8161 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     8221 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg
     8281 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     8341 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     8401 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     8461 tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa
     8521 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     8581 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     8641 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     8701 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     8761 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     8821 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     8881 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     8941 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     9001 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     9061 ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc
     9121 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     9181 acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc
     9241 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
     9301 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
     9361 acccttttag tcagtgtgga aaatctctag cagtacgtat agtagttcat gtcatcttat
     9421 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat
     9481 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     9541 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     9601 gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     9661 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     9721 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca
     9781 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg
     9841 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     9901 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     9961 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
    10021 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
    10081 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
    10141 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
    10201 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
    10261 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
    10321 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
    10381 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
    10441 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
    10501 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
    10561 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
    10621 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
    10681 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
    10741 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
    10801 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
    10861 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
    10921 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
    10981 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
    11041 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
    11101 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
    11161 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
    11221 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
    11281 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
    11341 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
    11401 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
    11461 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
    11521 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
    11581 aggatcttc
//
