LOCUS       sequence-308810-       12797 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of SpCas9; P2A site provides mKate2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12797)
  AUTHORS   Hanna RE, Hegde M, Fagre CR, DeWeirdt PC, Sangree AK, Szegletes Z, 
            Griffith A, Feeley MN, Sanson KR, Baidi Y, Koblan LW, Liu DR, Neal 
            JT, Doench JG
  TITLE     Massively parallel assessment of human variants with base editor 
            screens.
  JOURNAL   Cell. 2021 Feb 18;184(4):1064-1080.e20. doi: 
            10.1016/j.cell.2021.01.012.
  PUBMED    33606977
REFERENCE   2  (bases 1 to 12797)
  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"; doi: 
            "10.1016/j.cell.2021.01"; journalName: "Cell"; date: "2021-02-18- 
            18"; volume: "184"; issue: "4"; pages: "1064-1080.e20"
COMMENT     Sequence Label: pRDA_085
FEATURES             Location/Qualifiers
     source          1..12797
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        87..1265
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          318..1256
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1213..1233
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1282..1302
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1309..5409
                     /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             1309..5409
                     /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             5416..5463
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5476..5529
                     /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             5536..5931
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             5932..5988
                     /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             5995..6693
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     misc_feature    6712..7300
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6765..6785)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7183..7194)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7372..7605
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7683..7804
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7720..7739)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7774..7793
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7844..7979
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7999..8018)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8000..8018)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8028..8045)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8028..8044)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8037..8059)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8186..8641
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8273..8292)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8483..8504
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        8667..8771
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8772..8840,8841..9632)
                     /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     complement(8990..9009)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9803..10391
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10292..10311
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10545..10562
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    10679..10700
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(10715..10720,10721..10738,10739..10745)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10753..10769
                     /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)."
     primer_bind     10758..10780
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     10777..10793
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10777..10793
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     10812..10832
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        10814..10832
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        10860..11086
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             11087..11267
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11314..11439
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    11932..12165
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12350..12394
                     /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    12628..12745
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 cccgggtgca aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct
       61 tctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat
      121 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa
      181 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
      241 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
      301 ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg
      361 gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg attcttgatc
      421 ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt
      481 cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt
      541 ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat
      601 gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc
      661 acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca
      721 catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc
      781 aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg
      841 cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc
      901 ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac
      961 ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt
     1021 accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag
     1081 gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag
     1141 ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat
     1201 cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt
     1261 cgtgagggat ccaccatggc accaaagaag aagcggaagg tcgcagccga caagaagtac
     1321 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac
     1381 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag
     1441 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag
     1501 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc
     1561 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc
     1621 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag
     1681 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc
     1741 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg
     1801 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc
     1861 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc
     1921 gtagacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg
     1981 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc
     2041 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag
     2101 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag
     2161 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc
     2221 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac
     2281 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag
     2341 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc
     2401 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc
     2461 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcgtacgttc
     2521 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg
     2581 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc
     2641 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg
     2701 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc
     2761 gctagcgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag
     2821 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc
     2881 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa
     2941 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa
     3001 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat
     3061 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac
     3121 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg
     3181 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac
     3241 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag
     3301 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc
     3361 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa
     3421 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc
     3481 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac
     3541 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga
     3601 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa
     3661 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag
     3721 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac
     3781 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc
     3841 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc
     3901 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag
     3961 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga
     4021 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg
     4081 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag
     4141 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat
     4201 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac
     4261 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa
     4321 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc
     4381 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt
     4441 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca
     4501 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa
     4561 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc
     4621 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac
     4681 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg
     4741 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg
     4801 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag
     4861 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag
     4921 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa
     4981 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg
     5041 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac
     5101 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct
     5161 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag
     5221 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag
     5281 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc
     5341 accctgatcc accagagcat caccggcctg tacgagacac gaatcgattt gtctcagctg
     5401 ggaggcgacg ccgcaaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa
     5461 aaggcaggct caggcgaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat
     5521 cctggcccaa gcgctatggc caagcctttg tctcaagaag aatccaccct cattgaaaga
     5581 gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct
     5641 ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct
     5701 tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt
     5761 atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag
     5821 gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg
     5881 acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg cgcaacaaac
     5941 ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgac tagtatggtg
     6001 agcgagctga ttaaggagaa catgcacatg aagctataca tggagggcac cgtgaacaac
     6061 caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg
     6121 agaatcaagg cggtcgaggg cggccctctc cccttcgcct tcgacatcct ggctaccagc
     6181 ttcatgtacg gcagcaaaac cttcatcaac cacacccagg gcatccccga cttctttaag
     6241 cagtccttcc ccgagggctt cacatgggag agagtcacca catacgaaga cgggggcgtg
     6301 ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaagatc
     6361 agaggggtga acttcccatc caacggccct gtgatgcaga agaaaacact cggctgggag
     6421 gcctccaccg agaccctgta ccccgctgac ggcggcctgg aaggcagagc cgacatggcc
     6481 ctgaagctcg tgggcggggg ccacctgatc tgcaacttga agaccacata cagatccaag
     6541 aaacccgcta agaacctcaa gatgcccggc gtctactatg tggacagaag actggaaaga
     6601 atcaaggagg ccgacaaaga gacctacgtc gagcagcacg aggtggctgt ggccagatac
     6661 tgcgacctcc ctagcaaact ggggcacaga tgatagacgc gttaagtcga caatcaacct
     6721 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     6781 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     6841 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     6901 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     6961 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     7021 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     7081 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     7141 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     7201 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     7261 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
     7321 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     7381 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
     7441 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     7501 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     7561 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt
     7621 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
     7681 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     7741 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     7801 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc
     7861 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     7921 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     7981 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     8041 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     8101 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     8161 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     8221 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     8281 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     8341 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     8401 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     8461 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     8521 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     8581 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     8641 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     8701 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     8761 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     8821 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     8881 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     8941 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     9001 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     9061 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     9121 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     9181 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     9241 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     9301 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     9361 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     9421 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     9481 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     9541 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     9601 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     9661 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     9721 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     9781 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     9841 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     9901 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     9961 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
    10021 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
    10081 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
    10141 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
    10201 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
    10261 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
    10321 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
    10381 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
    10441 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
    10501 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
    10561 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
    10621 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
    10681 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
    10741 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
    10801 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat
    10861 gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc
    10921 cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg
    10981 tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc
    11041 gcattgcaga gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt
    11101 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
    11161 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
    11221 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
    11281 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
    11341 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
    11401 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
    11461 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
    11521 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
    11581 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
    11641 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
    11701 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
    11761 accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa
    11821 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
    11881 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
    11941 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
    12001 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
    12061 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
    12121 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
    12181 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
    12241 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
    12301 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
    12361 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
    12421 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
    12481 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
    12541 gacccacctc ccaaccccga ggggaccctg gtacccatgc atgcatgaat tctagatctt
    12601 gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
    12661 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
    12721 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg
    12781 gcgccggctc gaggggg
//
