LOCUS       sequence-329453-       13013 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses Cas9 and a non-targeting control guide RNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13013)
  AUTHORS   Hatterschide J, Bohidar AE, Grace M, Nulton TJ, Kim HW, Windle B, 
            Morgan IM, Munger K, White EA
  TITLE     PTPN14 degradation by high-risk human papillomavirus E7 limits 
            keratinocyte differentiation and contributes to HPV-mediated 
            oncogenesis.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Apr 2;116(14):7033-7042. doi: 
            10.1073/pnas.1819534116. Epub 2019 Mar 20.
  PUBMED    30894485
REFERENCE   2  (bases 1 to 13013)
  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: 
            "10.1073/pnas.1819534116"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2019-04-2- 2"; volume: "116"; issue: "14"; pages: "7033-7042"
COMMENT     Sequence Label: p8103 LentiCRISPR v2 sgNT-2
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     53..74
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(193..213)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        198..527
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      378..513
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     440..459
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        575..622
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             641..1015
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    1145..1266
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1182..1201)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1236..1255
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1315..1331)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1315..1331)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1328..1350)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1339..1355
                     /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(1363..1369,1370..1387,1388..1393))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1408..1429
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1546..1563)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1717..2305)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1797..1816)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2476..3267,3268..3336))
                     /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     3099..3118
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3337..3441)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3516..3535)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3707..4086
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4088..4286
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4241..4261
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4304..4484
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4531..4656
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5149..5382
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5567..5611
                     /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"
     misc_feature    5909..6026
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6077..6325
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6077..6097
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6248..6267
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6347..6422
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6484..6695
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6714..6723
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6720..10823
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6720..10823
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             10824..10871
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             10872..10895
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             10905..10961
                     /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             10962..11558
                     /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     11455..11475
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    11574..12162
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11627..11647)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12045..12056)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             12234..12467
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(12493..12510)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12499..12723
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(12769..13013,1..184)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12856..12875)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
       61 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
      121 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
      181 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
      241 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
      301 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
      361 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
      421 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
      481 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
      541 cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta
      601 tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc
      661 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct
      721 cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac
      781 cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg
      841 ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg
      901 ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc
      961 cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt
     1021 gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     1081 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     1141 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1201 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     1261 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     1321 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     1381 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     1441 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1501 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     1561 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     1621 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     1681 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     1741 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1801 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1861 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     1921 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     1981 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2041 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2101 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     2161 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2221 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2281 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2341 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2401 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2461 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     2521 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     2581 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     2641 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     2701 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     2761 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     2821 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     2881 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     2941 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3001 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3061 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3121 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3181 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3241 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3301 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     3361 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3421 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     3481 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3541 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     3601 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     3661 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
     3721 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     3781 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     3841 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     3901 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     3961 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     4021 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     4081 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     4141 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     4201 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     4261 gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc tctggttaga
     4321 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     4381 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     4441 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
     4501 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
     4561 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
     4621 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
     4681 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
     4741 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
     4801 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     4861 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     4921 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     4981 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     5041 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     5101 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     5161 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     5221 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     5281 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     5341 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     5401 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     5461 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     5521 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     5581 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     5641 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     5701 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     5761 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
     5821 gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct
     5881 gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca
     5941 gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa
     6001 aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt
     6061 ggttaattaa ggtaccgagg gcctatttcc catgattcct tcatatttgc atatacgata
     6121 caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca
     6181 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     6241 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     6301 tatcttgtgg aaaggacgaa acaccggatt gtggtcgctc aaaaccgttt tagagctaga
     6361 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     6421 gcttttttga attcgctagc taggtcttga aaggagtggg aattggctcc ggtgcccgtc
     6481 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
     6541 gatccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     6601 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     6661 ttctttttcg caacgggttt gccgccagaa cacaggaccg gttctagagc gctgccacca
     6721 tggacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga
     6781 tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc
     6841 acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg
     6901 ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct
     6961 atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac
     7021 tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca
     7081 acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga
     7141 aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca
     7201 tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg
     7261 tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca
     7321 tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac
     7381 ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc
     7441 tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg
     7501 atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc
     7561 agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc
     7621 tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta
     7681 tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc
     7741 agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg
     7801 gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg
     7861 aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga
     7921 agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg
     7981 ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg
     8041 agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca
     8101 gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag
     8161 tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga
     8221 acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt
     8281 ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga
     8341 gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg
     8401 tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct
     8461 ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta
     8521 tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc
     8581 tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc
     8641 acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca
     8701 ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg
     8761 atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca
     8821 gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc
     8881 acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag
     8941 tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga
     9001 tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa
     9061 tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg
     9121 tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg
     9181 atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata
     9241 tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg
     9301 acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga
     9361 actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga
     9421 ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc
     9481 tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca
     9541 ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca
     9601 agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact
     9661 accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt
     9721 accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga
     9781 tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca
     9841 acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc
     9901 ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg
     9961 ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc
    10021 agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg
    10081 ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct
    10141 attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga
    10201 aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact
    10261 ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt
    10321 actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc
    10381 agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc
    10441 actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac
    10501 agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga
    10561 tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc
    10621 ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc
    10681 ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag
    10741 aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg
    10801 acctgtctca gctgggaggc gacaagcgac ctgccgccac aaagaaggct ggacaggcta
    10861 agaagaagaa agattacaaa gacgatgacg ataagggatc cggcgcaaca aacttctctc
    10921 tgctgaaaca agccggagat gtcgaagaga atcctggacc gaccgagtac aagcccacgg
    10981 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
    11041 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
    11101 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
    11161 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
    11221 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
    11281 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
    11341 tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
    11401 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
    11461 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
    11521 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac
    11581 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
    11641 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
    11701 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
    11761 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
    11821 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
    11881 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
    11941 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg
    12001 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
    12061 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
    12121 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat
    12181 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
    12241 gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt
    12301 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
    12361 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
    12421 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa
    12481 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
    12541 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
    12601 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
    12661 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
    12721 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
    12781 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
    12841 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
    12901 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
    12961 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcg
//
