LOCUS       sequence-381521-       13494 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of eSpCas9 tagged with Zsgreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13494)
  AUTHORS   Mishra T, Bhardwaj V, Ahuja N, Gadgil P, Ramdas P, Shukla S, Chande 
            A
  TITLE     Improved loss-of-function CRISPR-Cas9 genome editing in human cells 
            concomitant with inhibition of TGF-beta signaling.
  JOURNAL   Mol Ther Nucleic Acids. 2022 Mar 8;28:202-218. doi: 
            10.1016/j.omtn.2022.03.003. eCollection 2022 Jun 14.
  PUBMED    35402072
REFERENCE   2  (bases 1 to 13494)
  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: "Mol Ther 
            Nucleic Acids."; date: "2022-03-8"; volume: "28:202-218. doi"; 
            pages: " 10.1016/j.omtn.2022.03.003. eCollection 2022 Jun 14"
COMMENT     Sequence Label: pScalps eSpCas9_Zsgreen gRNA scaffold
FEATURES             Location/Qualifiers
     source          1..13494
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    10..598
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(63..83)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(481..492)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1116..1296
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(1367..1955)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1447..1466)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2126..2917,2918..2986))
                     /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     2749..2768
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2987..3091)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3166..3185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3359..3379)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3364..3693
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3544..3679
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3606..3625
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        3705..4084
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4086..4284
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4239..4259
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4302..4482
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4529..4654
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5151..5384
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5568..5612
                     /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    5910..6027
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6078..6326
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6078..6098
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6249..6268
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8208..8283
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        8345..8556
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      8575..8584
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(8581..11121,11122..11124,11125..11586,11587..11589,
                     11590..11757,11758..11760,11761..12684)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             12685..12732
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             12742..12798
                     /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             12799..13494
                     /codon_start=1
                     /product="Zoanthus green fluorescent protein"
                     /label=ZsGreen1
                     /note="mammalian codon-optimized"
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
ORIGIN
        1 gcgatcgcta atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
       61 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
      121 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
      181 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
      241 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
      301 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
      361 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca
      421 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
      481 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
      541 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgctt
      601 aatcgcgtcg agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca
      661 atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac
      721 ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
      781 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc
      841 tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag
      901 ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga
      961 aggtagaaga agccaatgaa ggagagaaca cccgcttgtt acaccctgtg agcctgcatg
     1021 ggatggatga cccggagaga gaagtattag agtggaggtt tgacagccgc ctagcatttc
     1081 atcacatggc ccgagagctg catccggact gtactgggtc tctctggtta gaccagatct
     1141 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     1201 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     1261 tcagaccctt ttagtcagtg tggaaaatct ctagcagggc ccgtttcatg tgagcaaaag
     1321 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     1381 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     1441 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     1501 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     1561 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     1621 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     1681 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     1741 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     1801 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     1861 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     1921 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     1981 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     2041 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     2101 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     2161 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     2221 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     2281 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     2341 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     2401 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     2461 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     2521 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     2581 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     2641 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     2701 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     2761 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     2821 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     2881 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     2941 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     3001 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     3061 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     3121 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     3181 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     3241 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
     3301 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgctgtgg
     3361 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3421 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3481 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3541 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3601 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     3661 ggaggctttt ttggaggcct aggcttttgc aaaaagctcg cgttgacatt gattattgac
     3721 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     3781 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     3841 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     3901 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
     3961 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     4021 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     4081 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     4141 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     4201 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     4261 acggtgggag gtctatataa gcagcgcgtt ttgcctgtac tgggtctctc tggttagacc
     4321 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     4381 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     4441 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     4501 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     4561 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     4621 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     4681 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     4741 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     4801 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     4861 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     4921 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     4981 cgcacagcaa gcggccggcc gctgatcttc agacctggag gaggagatat gagggacaat
     5041 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     5101 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     5161 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     5221 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     5281 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     5341 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     5401 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     5461 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaatt aacaattaca
     5521 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag
     5581 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc
     5641 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt
     5701 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga
     5761 cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag
     5821 agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt gcgccaattc
     5881 tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac
     5941 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa
     6001 aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag agatccagtt
     6061 tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg catatacgat
     6121 acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag atattagtac
     6181 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     6241 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     6301 atatcttgtg gaaaggacga aacaccggag acggttgtaa atgagcacac aaaatacaca
     6361 tgctaaaata ttatattcta tgacctttat aaaatcaacc aaaatcttct ttttaataac
     6421 tttagtatca ataattagaa tttttatgtt cctttttgca aacttttaat aaaaatgagc
     6481 aaaataaaaa aacgctagtt ttagtaactc gcgttgtttt cttcaccttt aataatagct
     6541 actccaccac ttgttcctaa gcggtcagct cctgcttcaa tcattttttg agcatcttca
     6601 aatgttctaa ctccaccagc tgctttaact aaagcattgt ctttaacaac tgacttcatt
     6661 agtttaacat cttcaaatgt tgcacctgat tttgaaaatc ctgttgatgt tttaacaaat
     6721 tctaatccag cttcaacagc tatttcacaa gctttcatga tttcttcttt tgttaataaa
     6781 caattttcca taatacattt aacaacatgt gatccagctg ctttttttac agctttcatg
     6841 tcttctaaaa ctaattcata atttttgtct tttaatgcac caatatttaa taccatatca
     6901 atttctgttg caccatcttt aattgcttca gaaacttcga atgcttttgt agctgttgtg
     6961 catgcaccta gaggaaaacc tacaacattt gttattccta catttgtgcc ttttaataat
     7021 tctttacaat agcttgttca atatgaatta acacaaactg ttgcaaaatc aaattcaatt
     7081 gcttcatcac ataattgttt aatttcagct ttcgtagcat cttgttttaa taatgtgtga
     7141 tctatatatt tgtttagttt cattttttct cctatatatt catttttaat tttaattctt
     7201 taataatttc gtctacttta actttagcgt tttgaacaga ttcaccaaca cctataaaat
     7261 aaatttttag tttaggttca gttccacttg ggcgaacagc aaatcatgac ttatcttcta
     7321 aataaaattt tagtaagtct tgtcctggca tattatacat tccatcgatg tagtcttcaa
     7381 cattaacaac tttaagtcca gcaatttgag ttaagggtgt tgctctcaat gatttcatta
     7441 atggttcaat ttttaatttc ttttcttctg gtttaaaatt caagtttaaa gtgaaagtgt
     7501 aatatgcacc catttcttta aataaatctt ctaaatagtc tactaatgtt ttattttgtt
     7561 ttttataaaa tcaagcagcc tctgctatta atatagaagc ttgtattcca tctttatctc
     7621 tagctgagtc atcaattaca tatccataac tttcttcata agcaaaaaca aaatttaatc
     7681 cgttatcttc ttctttagca atttctctac ccattcattt aaatccagtt aaagttttta
     7741 caatattaac tccatatttt tcatgagcga ttctatcacc caaatcactt gttacaaaac
     7801 ttgaatatag agccggattt tttggaatgc tatttaagcg ttttagattt gataattttc
     7861 aatcaattaa aattggtcct gtttgatttc catctaatct tacaaaatga ccatcatgtt
     7921 ttattgccat tccaaatctg tcagcatctg ggtcattcat aataataata tctgcatcat
     7981 gtttaatacc atattcaagc ggtatttttc atgcaggatc aaattctgga tttggattta
     8041 caacattttt aaatgtttca tcttcaaatg catgctcttc aacctcaata acgttatatc
     8101 ctgattcacg taatattttt ggggtaaatt tagttcctgt tccattaact gcgctaaaaa
     8161 taatttttaa atctttttta gcttcttgct cttttttgta cgtctctgtt ttagagctag
     8221 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     8281 tgcttttttg aattcgctag ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt
     8341 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat
     8401 tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg
     8461 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac
     8521 gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag cgctgccacc
     8581 atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg
     8641 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
     8701 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
     8761 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
     8821 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
     8881 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
     8941 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
     9001 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
     9061 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     9121 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     9181 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     9241 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac
     9301 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     9361 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     9421 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     9481 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     9541 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     9601 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     9661 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     9721 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     9781 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     9841 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     9901 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     9961 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
    10021 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
    10081 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
    10141 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
    10201 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
    10261 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
    10321 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
    10381 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
    10441 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
    10501 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
    10561 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
    10621 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
    10681 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
    10741 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
    10801 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
    10861 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
    10921 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
    10981 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
    11041 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat
    11101 atcgtgcctc agagctttct ggccgacgac tccatcgaca acaaggtgct gaccagaagc
    11161 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
    11221 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
    11281 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
    11341 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
    11401 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
    11461 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
    11521 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
    11581 taccctgcgc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
    11641 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
    11701 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaaggcg
    11761 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
    11821 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
    11881 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
    11941 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
    12001 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
    12061 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
    12121 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
    12181 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
    12241 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
    12301 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
    12361 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
    12421 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
    12481 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
    12541 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
    12601 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
    12661 gacctgtctc agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca
    12721 aaaaagaaaa agggatccgg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc
    12781 gaagagaatc ctggaccgat ggcccagtcc aagcacggcc tgaccaagga gatgaccatg
    12841 aagtaccgca tggagggctg cgtggacggc cacaagttcg tgatcaccgg cgagggcatc
    12901 ggctacccct tcaagggcaa gcaggccatc aacctgtgcg tggtggaggg cggccccttg
    12961 cccttcgccg aggacatctt gtccgccgcc ttcatgtacg gcaaccgcgt gttcaccgag
    13021 tacccccagg acatcgtcga ctacttcaag aactcctgcc ccgccggcta cacctgggac
    13081 cgctccttcc tgttcgagga cggcgccgtg tgcatctgca acgccgacat caccgtgagc
    13141 gtggaggaga actgcatgta ccacgagtcc aagttctacg gcgtgaactt ccccgccgac
    13201 ggccccgtga tgaagaagat gaccgacaac tgggagccct cctgcgagaa gatcatcccc
    13261 gtgcccaagc agggcatctt gaagggcgac gtgagcatgt acctgctgct gaaggacggt
    13321 ggccgcttgc gctgccagtt cgacaccgtg tacaaggcca agtccgtgcc ccgcaagatg
    13381 cccgactggc acttcatcca gcacaagctg acccgcgagg accgcagcga cgccaagaac
    13441 cagaagtggc acctgaccga gcacgccatc gcctccggct ccgccttgcc ctga
//
