LOCUS       sequence-232926-       11964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ubiquitin promoter expresses mCherry 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 11964)
  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 11964)
  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: pRubiC-T2A-Cas9
FEATURES             Location/Qualifiers
     source          1..11964
                     /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      3444..3453
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3450..4157
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPDDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3595..3613)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3863..3882
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4158..4211
                     /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             4218..4283
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4290..4310
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4335..8435
                     /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             4335..8435
                     /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             8436..8483
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     misc_feature    8511..9099
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8564..8584)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8982..8993)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(9102..9118)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9103..9119)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             9234..9659
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(9762..9784)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9920..9940)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9925..10252
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10102..10238
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10274..10293
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(10374..10391)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10545..11133)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10625..10644)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11304..11964,1..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     11927..11946
                     /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 cccgccacca tggtgagcaa gggcgaggag gataacatgg
     3481 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg
     3541 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     3601 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca
     3661 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
     3721 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     3781 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
     3841 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct
     3901 cctccgagcg gatgtacccc gatgacggcg ccctgaaggg cgagatcaag cagaggctga
     3961 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
     4021 ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg
     4081 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca
     4141 tggacgagct gtacaaggag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga
     4201 atcccggccc tgaattcgac tataaggacc acgacggaga ctacaaggat catgatattg
     4261 attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg
     4321 gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc aactctgtgg
     4381 gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca
     4441 acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg
     4501 aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga
     4561 accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct
     4621 tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc
     4681 ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc
     4741 acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg
     4801 ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg
     4861 acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg
     4921 aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga
     4981 gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc
     5041 tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg
     5101 acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca
     5161 acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt
     5221 ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc
     5281 tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag
     5341 ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga
     5401 acggctacgc cggctacatt gacggcggag ccagccagga agagttctac aagttcatca
     5461 agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg
     5521 acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg
     5581 gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc
     5641 gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca
     5701 ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga
     5761 acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca
     5821 acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt
     5881 acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc
     5941 ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc
     6001 ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact
     6061 ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc
     6121 tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg
     6181 aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga
     6241 aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca
     6301 ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca
     6361 agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga
     6421 tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg
     6481 gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca
     6541 tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg
     6601 agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca
     6661 gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga
     6721 aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc
     6781 agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg
     6841 atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc
     6901 tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga
     6961 agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt
     7021 tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca
     7081 tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact
     7141 cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca
     7201 ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg
     7261 agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc
     7321 tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg
     7381 acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact
     7441 tcttctacag caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga
     7501 tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg
     7561 gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa
     7621 agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg
     7681 ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc
     7741 ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac
     7801 tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga
     7861 atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca
     7921 agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg
     7981 ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt
     8041 acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc
     8101 tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct
     8161 ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc
     8221 accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca
     8281 atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca
     8341 ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg
     8401 agacacggat cgacctgtct cagctgggag gcgacaaaag gccggcggcc acgaaaaagg
     8461 ccggccaggc aaaaaagaaa aagtaagaat tcgatatcaa gcttatcgat aatcaacctc
     8521 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     8581 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     8641 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     8701 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     8761 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     8821 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     8881 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     8941 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     9001 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     9061 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgag
     9121 atatcagtgg tccaggctct agttttgact caacaatatc accagctgaa gcctatagag
     9181 tacgagccat agataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa
     9241 gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa
     9301 aatacataac tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagggtcg
     9361 accctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc
     9421 ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga
     9481 gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc
     9541 gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc
     9601 tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt
     9661 gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg
     9721 taagctggct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     9781 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     9841 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     9901 ggagtgtaga tccggctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     9961 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtgaaagt
    10021 ccccaggctc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
    10081 agtcccgccc ctactccgcc catcccgccc ctactccgcc cagttccgcc caattctccg
    10141 cccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
    10201 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagcttac
    10261 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
    10321 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
    10381 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
    10441 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
    10501 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
    10561 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
    10621 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
    10681 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
    10741 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
    10801 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
    10861 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
    10921 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
    10981 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
    11041 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
    11101 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
    11161 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
    11221 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
    11281 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
    11341 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
    11401 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
    11461 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
    11521 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
    11581 tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc
    11641 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
    11701 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
    11761 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
    11821 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
    11881 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca
    11941 catagcagaa ctttaaaagt gctc
//
