LOCUS       sequence-142640-       11985 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ubiquitin promoter expresses GFP and humanized spCas9 (PX330) via a 
            T2A motif. For cell transfection or use in retroviral (MMuLV) 
            packaging. Use Pac1 and/or BstB1 sites to insert sgRNAs from PXL..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11985)
  AUTHORS   Williams MR, Fricano-Kugler CJ, Getz SA, Skelton PD, Lee J, Rizzuto 
            CP, Geller JS, Li M, Luikart BW
  TITLE     A Retroviral CRISPR-Cas9 System for Cellular Autism-Associated 
            Phenotype Discovery in Developing Neurons.
  JOURNAL   Sci Rep. 2016 May 10;6:25611. doi: 10.1038/srep25611.
  PUBMED    27161796
REFERENCE   2  (bases 1 to 11985)
  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: "Sci Rep."; 
            date: "2016-05-10"; volume: "6:25611. doi"; pages: " 
            10.1038/srep25611"
COMMENT     Sequence Label: pRubiG-T2A-Cas9
FEATURES             Location/Qualifiers
     source          1..11985
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(201..305)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     373..391
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        491..870
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        871..1073
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1024..1044
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             1074..1249
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    1312..1511
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1712..2127
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     2057..2079
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     2095..2111
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        2198..3408
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3245..3264
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      3456..3465
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3462..3464,3465..3467,3468..4178)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3462..3464,3465..3467,3468..4178)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3768..3787)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4115..4136
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4179..4232
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4239..4304
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4311..4331
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4356..8456
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4356..8456
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             8457..8504
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     misc_feature    8532..9120
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8585..8605)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9003..9014)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(9123..9139)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9124..9140)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             9255..9680
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(9783..9805)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9941..9961)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9946..10273
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10123..10259
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10295..10314
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(10395..10412)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10566..11154)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10646..10665)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11325..11985,1..131,132..200))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     11948..11967
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
       61 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
      121 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
      181 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
      241 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
      301 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
      361 ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt agcttggcca
      421 ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg tccaacatta
      481 ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      541 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      601 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      661 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      721 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      781 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      841 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      901 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      961 agtttgtttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     1021 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcaataaa
     1081 agagcccaca acccctcact cggcgcgcca gtcttccgat agactgcgtc gcccgggtac
     1141 ccgtattccc aataaagcct cttgctgttt gcatccgaat cgtggtctcg ctgttccttg
     1201 ggagggtctc ctctgagtga ttgactaccc acgacggggg tctttcattt gggggctcgt
     1261 ccgggatttg gagacccctg cccagggacc accgacccac caccgggagg taagctggcc
     1321 agcaacttat ctgtgtctgt ccgattgtct agtgtctatg tttgatgtta tgcgcctgcg
     1381 tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt
     1441 ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc gtttttgtgg
     1501 cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg tggttctggt
     1561 aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggaa
     1621 ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct
     1681 gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc ccttaagttt
     1741 gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa
     1801 gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg tcggatggcg
     1861 cgagacggca cctttaaccg aggactcatc acccaggtta agatcaaggt cttttcacct
     1921 ggcccgcatg gacacccaga ccaggtcccg tacatcgtga cctgggaagc cttggctttt
     1981 gacccccctc cctgggtcaa gccctttgta caccctaagc ctccgcctcc tcttcctcca
     2041 tccgccccgt ctctccccct tgaacctcct cgttcgaccc cgcctcgatc ctccctttat
     2101 ccagccctca ctccttctct aggcgccgga attgaagatc tcacacagcg gccgcacaca
     2161 cttcgaacct taattaaccc gtgtcggctc cagatctggc ctccgcgccg ggttttggcg
     2221 cctcccgcgg gcgcccccct cctcacggcg agcgctgcca cgtcagacga agggcgcagc
     2281 gagcgtcctg atccttccgc ccggacgctc aggacagcgg cccgctgctc ataagactcg
     2341 gccttagaac cccagtatca gcagaaggac attttaggac gggacttggg tgactctagg
     2401 gcactggttt tctttccaga gagcggaaca ggcgaggaaa agtagtccct tctcggcgat
     2461 tctgcggagg gatctccgtg gggcggtgaa cgccgatgat tatataagga cgcgccgggt
     2521 gtggcacagc tagttccgtc gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc
     2581 tgtgatcgtc acttggtgag tagcgggctg ctgggctggc cggggctttc gtggccgccg
     2641 ggccgctcgg tgggacggaa gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc
     2701 gagcaaggtt gccctgaact gggggttggg gggagcgcac aaaatggcgg ctgttcccga
     2761 gtcttgaatg gaagacgctt gtaaggcggg ctgtgaggtc gttgaaacaa ggtggggggc
     2821 atggtgggcg gcaagaaccc aaggtcttga ggccttcgct aatgcgggaa agctcttatt
     2881 cgggtgagat gggctggggc accatctggg gaccctgacg tgaagtttgt cactgactgg
     2941 agaactcggg tttgtcgtct ggttgcgggg gcggcagtta tgcggtgccg ttgggcagtg
     3001 cacccgtacc tttgggagcg cgcgcctcgt cgtgtcgtga cgtcacccgt tctgttggct
     3061 tataatgcag ggtggggcca cctgccggta ggtgtgcggt aggcttttct ccgtcgcagg
     3121 acgcagggtt cgggcctagg gtaggctctc ctgaatcgac aggcgccgga cctctggtga
     3181 ggggagggat aagtgaggcg tcagtttctt tggtcggttt tatgtaccta tcttcttaag
     3241 tagctgaagc tccggttttg aactatgcgc tcggggttgg cgagtgtgtt ttgtgaagtt
     3301 ttttaggcac cttttgaaat gtaatcattt gggtcaatat gtaattttca gtgttagact
     3361 agtaaattgt ccgctaaatt ctggccgttt ttggcttttt tgttagacga agcttgggct
     3421 gcaggtcgac tctagaggat ccccgggtac cggtcgccac catggtgagc aagggcgagg
     3481 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     3541 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     3601 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     3661 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     3721 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     3781 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     3841 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     3901 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     3961 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     4021 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     4081 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     4141 ccgccgggat cactctcggc atggacgagc tgtacaagga gggcagagga agtcttctaa
     4201 catgcggtga cgtggaggag aatcccggcc ctgaattcga ctataaggac cacgacggag
     4261 actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga
     4321 agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc atcggcctgg
     4381 acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca
     4441 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag
     4501 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa
     4561 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga
     4621 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg
     4681 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg
     4741 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg
     4801 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga
     4861 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc
     4921 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg
     4981 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc
     5041 ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc
     5101 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca
     5161 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt
     5221 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca
     5281 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc
     5341 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga
     5401 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg
     5461 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc
     5521 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca
     5581 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt
     5641 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct
     5701 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg
     5761 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga
     5821 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca
     5881 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg
     5941 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg
     6001 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca
     6061 agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct
     6121 ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg
     6181 aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag
     6241 agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc
     6301 agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca
     6361 tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca
     6421 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga
     6481 aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca
     6541 gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag
     6601 tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca
     6661 cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag
     6721 agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga
     6781 agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca
     6841 tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg
     6901 actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg
     6961 tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca
     7021 agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg
     7081 aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc
     7141 acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga
     7201 tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt
     7261 tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga
     7321 acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt
     7381 acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg
     7441 gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga
     7501 ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg
     7561 gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc
     7621 cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta
     7681 tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga
     7741 agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg
     7801 aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg
     7861 aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag
     7921 tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc
     7981 ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct
     8041 ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg
     8101 aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca
     8161 tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag
     8221 tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca
     8281 tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca
     8341 ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc
     8401 agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaaaa
     8461 ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtaagaa ttcgatatca
     8521 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     8581 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     8641 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     8701 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     8761 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     8821 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     8881 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     8941 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     9001 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     9061 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     9121 atcgataccg tcgacctcga gatatcagtg gtccaggctc tagttttgac tcaacaatat
     9181 caccagctga agcctataga gtacgagcca tagataaaat aaaagatttt atttagtctc
     9241 cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg
     9301 ccattttgca aggcatggaa aaatacataa ctgagaatag agaagttcag atcaaggtca
     9361 ggaacagatg gaacagggtc gaccctagag aaccatcaga tgtttccagg gtgccccaag
     9421 gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt
     9481 tcgcgcgctt ctgctccccg agctcaataa aagagcccac aacccctcac tcggggcgcc
     9541 agtcctccga ttgactgagt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt
     9601 gcatccgact tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc
     9661 gtcagcgggg gtctttcatt tgggggctcg tccgggatcg ggagacccct gcccagggac
     9721 caccgaccca ccaccgggag gtaagctggc tgcctcgcgc gtttcggtga tgacggtgaa
     9781 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     9841 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     9901 acccagtcac gtagcgatag cggagtgtag atccggctgt ggaatgtgtg tcagttaggg
     9961 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
    10021 tcagcaacca ggtgtgaaag tccccaggct cccagcaggc agaagtatgc aaagcatgca
    10081 tctcaattag tcagcaacca tagtcccgcc cctactccgc ccatcccgcc cctactccgc
    10141 ccagttccgc ccaattctcc gcccccatgg ctgactaatt ttttttattt atgcagaggc
    10201 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
    10261 aggcttttgc aaaaagctta ctggcttaac tatgcggcat cagagcagat tgtactgaga
    10321 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
    10381 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
    10441 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
    10501 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
    10561 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
    10621 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
    10681 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
    10741 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
    10801 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
    10861 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
    10921 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
    10981 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
    11041 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
    11101 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
    11161 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
    11221 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
    11281 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
    11341 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
    11401 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
    11461 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
    11521 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
    11581 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
    11641 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
    11701 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
    11761 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
    11821 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
    11881 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
    11941 acacgggata ataccgcgcc acatagcaga actttaaaag tgctc
//
