LOCUS       sequence-417774-       13703 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral tricistronic complex expressing Cas9, mNeonGreen, and 
            blasticidin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13703)
  AUTHORS   Teh SSK, Bowland K, Halper-Stromberg E, Kotwal A, Bennett A, Skaist 
            A, Tang J, Cai F, Macoretta A, Liang H, Kamiyama H, Wheelan S, Lin 
            MT, Hruban RH, Hung CF, Goldstein M, Scharpf RB, Roberts NJ, 
            Eshleman JR
  TITLE     CRISPR-Cas9 for selective targeting of somatic mutations in 
            pancreatic cancers.
  JOURNAL   NAR Cancer. 2024 Jun 19;6(2):zcae028. doi: 10.1093/narcan/zcae028. 
            eCollection 2024 Jun.
  PUBMED    38915758
REFERENCE   2  (bases 1 to 13703)
  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: "NAR 
            Cancer."; date: "2024-06-19"; volume: "6(2):zcae028. doi"; pages: " 
            10.1093/narcan/zcae028. eCollection 2024 Jun"
COMMENT     Sequence Label: pLenti_Cas9_T2A_mNeonGreen_P2A_blasticidin
FEATURES             Location/Qualifiers
     source          1..13703
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             69..125
                     /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             126..524
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    549..1137
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(602..622)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1020..1031)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1140..1156)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1141..1157)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             1662..1842
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(1868..1885)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1874..2098
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2144..2572
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2231..2250)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2441..2462
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2581..2601)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2746..2880
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2807..2826
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2942..2989
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3008..3382
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3512..3633
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3549..3568)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3603..3622
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3682..3698)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3682..3698)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3695..3717)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3706..3722
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3730..3736,3737..3754,3755..3760))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3775..3796
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3913..3930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4084..4672)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4164..4183)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4843..5634,5635..5703))
                     /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     5466..5485
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5704..5808)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5883..5902)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6074..6453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6455..6653
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6608..6628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6671..6851
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6898..7023
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7516..7749
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7934..7978
                     /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    8276..8393
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8486..8697
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      8705..8714
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             8714..8779
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             8786..8806
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8831..12931
                     /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             8831..12931
                     /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             12932..12979
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             12986..13039
                     /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             join(13040..13703,1..41)
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      13292..13301
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 aggcctttac cgatgtgatg ggcatggacg agctgtacaa gtctagaagc gctggtaccg
       61 gatccggcgc aacaaacttc tctctgctga aacaagccgg agatgtcgaa gagaatcctg
      121 gaccgatggc caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta
      181 caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg
      241 acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac
      301 tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga
      361 tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg
      421 atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg
      481 ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaagaattc gatatcaagc
      541 ttatcggtaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
      601 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
      661 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
      721 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
      781 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
      841 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
      901 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
      961 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     1021 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     1081 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     1141 gataccgtcg acctcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag
     1201 ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag
     1261 tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact
     1321 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatcc
     1381 ttgatctgtg gatctaccac acacaaggct acttccctga ttggcagaac tacacaccag
     1441 ggccagggat cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc
     1501 aagagaaggt agaagaagcc aatgaaggag agaacacccg cttgttacac cctgtgagcc
     1561 tgcatgggat ggatgacccg gagagagaag tattagagtg gaggtttgac agccgcctag
     1621 catttcatca catggcccga gagctgcatc cggactgtac tgggtctctc tggttagacc
     1681 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     1741 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     1801 gatccctcag acccttttag tcagtgtgga aaatctctag cagggcccgt ttaaacccgc
     1861 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     1921 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     1981 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2041 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2101 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2161 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2221 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2281 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2341 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2401 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2461 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2521 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     2581 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2641 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     2701 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     2761 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     2821 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     2881 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg
     2941 tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa
     3001 ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg
     3061 agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt
     3121 cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt
     3181 gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg
     3241 gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg
     3301 cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt
     3361 cgtggccgag gagcaggact gacacgtgct acgagatttc gattccaccg ccgccttcta
     3421 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3481 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     3541 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3601 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     3661 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3721 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3781 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3841 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3901 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3961 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4021 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4081 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4141 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4201 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4261 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4321 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4381 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4441 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4501 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4561 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4621 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4681 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4741 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4801 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4861 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4921 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4981 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5041 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5101 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5161 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5221 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5281 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5341 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5401 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5461 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5521 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5581 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5641 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5701 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5761 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5821 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     5881 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     5941 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     6001 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     6061 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
     6121 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     6181 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     6241 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     6301 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     6361 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     6421 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     6481 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     6541 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     6601 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagcgcgttt
     6661 tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     6721 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     6781 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     6841 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
     6901 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
     6961 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
     7021 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
     7081 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
     7141 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
     7201 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
     7261 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
     7321 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc
     7381 tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa
     7441 aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa
     7501 aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat
     7561 gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca
     7621 gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt
     7681 ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca
     7741 acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg
     7801 gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga
     7861 gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca
     7921 aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg
     7981 gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg
     8041 aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca
     8101 gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc
     8161 cgaaggaata gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa
     8221 cggatcggca ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta
     8281 aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa
     8341 cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt
     8401 attacaggga cagcagagat ccagtttggt taattagcta gctaggtctt gaaaggagtg
     8461 ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     8521 ttggggggag gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg
     8581 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     8641 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggac
     8701 cggtgccacc atggactata aggaccacga cggagactac aaggatcatg atattgatta
     8761 caaagacgat gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt
     8821 cccagcagcc gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg
     8881 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
     8941 cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac
     9001 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
     9061 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
     9121 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
     9181 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
     9241 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
     9301 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
     9361 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
     9421 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
     9481 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
     9541 cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
     9601 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
     9661 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
     9721 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
     9781 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
     9841 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
     9901 ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc
     9961 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct
    10021 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga
    10081 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga
    10141 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
    10201 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
    10261 cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt
    10321 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt
    10381 caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc
    10441 cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa
    10501 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
    10561 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct
    10621 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga
    10681 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac
    10741 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg
    10801 ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
    10861 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca
    10921 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga
    10981 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct
    11041 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa
    11101 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
    11161 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga
    11221 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa
    11281 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt
    11341 ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac
    11401 cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
    11461 gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga
    11521 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa
    11581 gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg
    11641 gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct
    11701 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
    11761 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat
    11821 caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt
    11881 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt
    11941 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
    12001 gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg
    12061 ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac
    12121 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa
    12181 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac
    12241 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
    12301 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
    12361 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
    12421 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
    12481 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
    12541 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
    12601 tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa
    12661 gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg
    12721 ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
    12781 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag
    12841 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
    12901 acggatcgac ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg
    12961 ccaggcaaaa aagaaaaagg aattcgaggg cagaggaagt ctgctaacat gcggtgacgt
    13021 cgaggagaat cctggcccag tgagcaaggg cgaggaggat aacatggcct ctctcccagc
    13081 gacacatgag ttacacatct ttggctccat caacggtgtg gactttgaca tggtgggtca
    13141 gggcaccggc aatccaaatg atggttatga ggagttaaac ctgaagtcca ccaagggtga
    13201 cctccagttc tccccctgga ttctggtccc tcatatcggg tatggcttcc atcagtacct
    13261 gccctaccct gacgggatgt cgcctttcca ggccgccatg gtagatggct ccggatacca
    13321 agtccatcgc acaatgcagt ttgaagatgg tgcctccctt actgttaact accgctacac
    13381 ctacgaggga agccacatca aaggagaggc ccaggtgaag gggactggtt tccctgctga
    13441 cggtcctgtg atgaccaact cgctgaccgc tgcggactgg tgcaggtcga agaagactta
    13501 ccccaacgac aaaaccatca tcagtacctt taagtggagt tacaccactg gaaatggcaa
    13561 gcgctaccgg agcactgcgc ggaccaccta cacctttgcc aagccaatgg cggctaacta
    13621 tctgaagaac cagccgatgt acgtgttccg taagacggag ctcaagcact ccaagaccga
    13681 gctcaacttc aaggagtggc aaa
//
