LOCUS       sequence-487500-       14735 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Doxycycline inducible CRISPRgenee construct with a blasticidin 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14735)
  AUTHORS   Stadager J, Bernardini C, Hartmann L, May H, Wiepcke J, Kuban M, 
            Najafova Z, Johnsen SA, Legewie S, Traube FR, Jude J, Rathert P
  TITLE     CRISPR GENome and epigenome engineering improves loss-of-function 
            genetic-screening approaches.
  JOURNAL   Cell Rep Methods. 2025 Jun 16;5(6):101078. doi: 
            10.1016/j.crmeth.2025.101078. Epub 2025 Jun 10.
  PUBMED    40499551
REFERENCE   2  (bases 1 to 14735)
  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: "Cell Rep 
            Methods."; date: "2025-06-16"; volume: "5(6):101078. doi"; pages: " 
            10.1016/j.crmeth.2025.101078. Epub 2025 Jun 10"
COMMENT     Sequence Label: TRE3G-ZIM3-Cas9-P2A-GFP-PGK-Blasti
FEATURES             Location/Qualifiers
     source          1..14735
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      142..570
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(229..248)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     439..460
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(579..599)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        584..913
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      764..899
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     826..845
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        961..1008
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1027..1401
                     /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    1531..1652
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1568..1587)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1622..1641
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1701..1717)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1701..1717)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1714..1736)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1725..1741
                     /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(1749..1755,1756..1773,1774..1779))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1794..1815
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1932..1949)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2103..2691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2183..2202)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2862..3653,3654..3722))
                     /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     3485..3504
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3723..3827)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3902..3921)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4093..4472
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4474..4672
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4627..4647
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4690..4870
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4917..5042
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5535..5768
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5953..5997
                     /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    6295..6412
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6476..6854
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    6484..6502
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6520..6538
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6556..6574
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6592..6610
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6628..6646
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6664..6682
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6700..6718
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     6763..6787
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(7182..7206)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             7226..7246
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      7301..7310
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7307..11410
                     /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             7307..11410
                     /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             11411..11458
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             11459..11482
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             11498..11554
                     /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             join(11555..11557,11558..11560,11561..12274)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(11861..11880)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     12208..12229
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        12293..12792
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(12328..12351)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     12725..12744
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     misc_feature    13282..13870
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(13335..13355)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(13753..13764)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(13873..13889)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13874..13890)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             14395..14575
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(14601..14618)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(14607..14735,1..96)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
       61 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
      121 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
      181 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
      241 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
      301 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
      361 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
      421 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
      481 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
      541 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
      601 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
      661 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
      721 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
      781 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
      841 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
      901 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcagcacgt
      961 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
     1021 taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga
     1081 gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc
     1141 gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg
     1201 ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg
     1261 tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc
     1321 gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc
     1381 gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat
     1441 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     1501 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     1561 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     1621 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     1681 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     1741 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     1801 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1861 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1921 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1981 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2041 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2101 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2161 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2221 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2281 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2341 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2401 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2461 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2521 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2581 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2641 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2701 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2761 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2821 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2881 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2941 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3001 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3061 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3121 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3181 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3241 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3301 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3361 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3421 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3481 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3541 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3601 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3661 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3721 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3781 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3841 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     3901 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     3961 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     4021 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     4081 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     4141 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     4201 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     4261 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     4321 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     4381 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     4441 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     4501 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     4561 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     4621 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt
     4681 gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     4741 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     4801 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     4861 atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct
     4921 ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
     4981 tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg
     5041 tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg
     5101 gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg
     5161 cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac
     5221 aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc
     5281 tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag
     5341 aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct
     5401 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     5461 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     5521 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     5581 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     5641 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     5701 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     5761 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     5821 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     5881 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     5941 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     6001 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     6061 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     6121 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc
     6181 gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac
     6241 ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa
     6301 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     6361 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     6421 ttacagggac agcagagatc cagtttggtt aattagctag cgatcctcta gattcgagtt
     6481 tactccctat cagtgataga gaacgtatga agagtttact ccctatcagt gatagagaac
     6541 gtatgcagac tttactccct atcagtgata gagaacgtat aaggagttta ctccctatca
     6601 gtgatagaga acgtatgacc agtttactcc ctatcagtga tagagaacgt atctacagtt
     6661 tactccctat cagtgataga gaacgtatat ccagtttact ccctatcagt gatagagaac
     6721 gtataagctt taggcgtgta cggtgggcgc ctataaaagc agagctcgtt tagtgaaccg
     6781 tcagatcgcc tggagcaatt ccacaacact tttgtcttat accaactttc cgtaccactt
     6841 cctaccctcg taaagtcgag cttgcgttgg atccaccagc atgaacaatt cccagggaag
     6901 agtgaccttc gaggatgtca ctgtgaactt cacccagggg gagtggcagc ggctgaatcc
     6961 cgaacagaga aacttgtaca gggatgtgat gctggagaat tacagcaacc ttgtctctgt
     7021 gggacaaggg gaaaccacca aacccgatgt gatcttgagg ttggaacaag gaaaggagcc
     7081 atggttggag gaagaggaag tgctgggaag tggccgtgca gaaaaaaatg gggacattgg
     7141 agggcagatt tggaagccaa aggatgtgaa agagagtctc tacccaactt tcttgtacaa
     7201 agtggttggt ggatctggag gttctcccaa gaagaagcgc aaggtgggac gcgtctgcag
     7261 gatatcaagc ttgcggtacc gcgggcccgg gatcagcgct gccaccatgg acaagaagta
     7321 cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
     7381 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
     7441 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa
     7501 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
     7561 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
     7621 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
     7681 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
     7741 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
     7801 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
     7861 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
     7921 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
     7981 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag
     8041 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
     8101 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
     8161 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
     8221 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     8281 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     8341 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
     8401 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     8461 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     8521 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     8581 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     8641 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     8701 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     8761 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
     8821 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
     8881 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     8941 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     9001 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     9061 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     9121 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     9181 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     9241 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     9301 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     9361 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
     9421 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     9481 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     9541 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     9601 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     9661 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     9721 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     9781 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag
     9841 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     9901 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     9961 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
    10021 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
    10081 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
    10141 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
    10201 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
    10261 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
    10321 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
    10381 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
    10441 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
    10501 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
    10561 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
    10621 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
    10681 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
    10741 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctactggg
    10801 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
    10861 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
    10921 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
    10981 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
    11041 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
    11101 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
    11161 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
    11221 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
    11281 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
    11341 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
    11401 gggaggcgac aagcgacctg ccgccacaaa gaaggctgga caggctaaga agaagaaaga
    11461 ttacaaagac gatgacgata agggatccgg cagcggcgcc accaacttca gcctgctgaa
    11521 gcaggccggc gacgtggagg agaaccccgg ccccatggtg agcaagggcg aggagctgtt
    11581 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
    11641 cgtgtctggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
    11701 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
    11761 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
    11821 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
    11881 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
    11941 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
    12001 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggcgaact tcaagatccg
    12061 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
    12121 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
    12181 caaagacccc aacgagaaac gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
    12241 gatcactctc ggcatggacg agctgtacaa gtaagaattc cgcaattcta ccgggtaggg
    12301 gaggcgcttt tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg
    12361 cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg
    12421 gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc
    12481 ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact
    12541 agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc
    12601 ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc
    12661 gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc
    12721 ccggcattct gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct
    12781 ccgggccttt cggacgtgga ggagaacccc ggccccatga agaccttcaa catctctcag
    12841 caggaccttg agctggtgga agttgccact gagaaaatca ccatgctcta tgaggacaac
    12901 aagcaccatg ttggtgctgc catcaggacc aagacaggag aaatcatctc tgctgtccac
    12961 attgaagcct acattggcag ggtcactgtg tgtgcagagg ccattgccat tgggtctgca
    13021 gtctccaatg ggcagaagga ctttgacacc attgtggctg tgaggcaccc ctacagtgat
    13081 gaggtggaca ggtccatcag agtggtgtcc ccctgtggca tgtgcaggga gctcatctca
    13141 gactatgccc ctgattgctt tgttctgatt gagatgaatg gcaagctggt caagacaacc
    13201 attgaggagc tgattccact gaaatacacc agaaactaat tcgaaagatc cgctagcgaa
    13261 ttcgatatca agcttatcgg taatcaacct ctggattaca aaatttgtga aagattgact
    13321 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
    13381 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
    13441 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
    13501 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
    13561 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
    13621 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
    13681 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
    13741 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
    13801 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
    13861 gcctccccgc atcgataccg tcgacctcga gacctagaaa aacatggagc aatcacaagt
    13921 agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga ggaggaggag
    13981 gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa ggcagctgta
    14041 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
    14101 agacaagata tccttgatct gtggatctac cacacacaag gctacttccc tgattggcag
    14161 aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg ctacaagcta
    14221 gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac ccgcttgtta
    14281 caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga gtggaggttt
    14341 gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg tactgggtct
    14401 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
    14461 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
    14521 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc
    14581 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
    14641 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
    14701 aaatgaggaa attgcatcgc attgtctgag taggt
//
