LOCUS       sequence-209972-       12470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This lentiviral construct delivers hSpCas9 and blasticidin S 
            resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12470)
  AUTHORS   Stringer BW, Day BW, D'Souza RCJ, Jamieson PR, Ensbey KS, Bruce ZC, 
            Lim YC, Goasdoue K, Offenhauser C, Akgul S, Allan S, Robertson T, 
            Lucas P, Tollesson G, Campbell S, Winter C, Do H, Dobrovic A, Inglis
            PL, Jeffree RL, Johns TG, Boyd AW
  TITLE     A reference collection of patient-derived cell line and xenograft 
            models of proneural, classical and mesenchymal glioblastoma.
  JOURNAL   Sci Rep. 2019 Mar 20;9(1):4902. doi: 10.1038/s41598-019-41277-z.
  PUBMED    30894629
REFERENCE   2  (bases 1 to 12470)
  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: "2019-03-20"; volume: "9(1):4902. doi"; pages: " 
            10.1038/s41598-019-41277-z"
COMMENT     Sequence Label: lenti-SpCas9 blast
FEATURES             Location/Qualifiers
     source          1..12470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        242..621
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        623..821
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     776..796
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             839..1019
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1066..1191
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /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    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2673..2884
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2903..2912
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2909..7012
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2909..7012
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7013..7060
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7061..7084
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7094..7150
                     /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             7151..7546
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    7562..8150
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7615..7635)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8033..8044)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8222..8455
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8481..8498)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8487..8711
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8757..9185
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8844..8863)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9054..9075
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9194..9214)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9199..9528
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9379..9514
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9441..9460
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9576..9623
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9642..10016
                     /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    10146..10267
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10183..10202)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10237..10256
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10316..10332)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10316..10332)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10329..10351)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10340..10356
                     /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(10364..10370,10371..10388,10389..10394))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10409..10430
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10547..10564)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10718..11306)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10798..10817)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11477..12268,12269..12337))
                     /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     12100..12119
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12338..12442)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgcgcgt
      241 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
      301 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      361 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      421 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      481 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      541 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      601 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      661 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      721 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      781 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg cctgtactgg
      841 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      901 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      961 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     1021 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
     1081 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
     1141 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
     1201 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
     1261 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
     1321 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
     1381 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
     1441 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
     1501 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta attaaggtac cctcgagtgt acagctagct aggtcttgaa
     2641 aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     2701 cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt ggcgcggggt
     2761 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     2821 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     2881 acaggaccgg ttctagagcg ctgccaccat ggacaagaag tacagcatcg gcctggacat
     2941 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     3001 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
     3061 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     3121 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     3181 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     3241 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     3301 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     3361 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     3421 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     3481 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     3541 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     3601 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     3661 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     3721 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     3781 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     3841 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     3901 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     3961 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     4021 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     4081 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     4141 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     4201 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     4261 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     4321 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     4381 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     4441 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     4501 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     4561 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     4621 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     4681 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     4741 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     4801 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     4861 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     4921 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     4981 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     5041 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     5101 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     5161 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     5221 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     5281 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     5341 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     5401 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc
     5461 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     5521 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     5581 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     5641 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     5701 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     5761 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     5821 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     5881 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     5941 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     6001 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     6061 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     6121 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     6181 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     6241 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     6301 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     6361 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     6421 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     6481 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     6541 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     6601 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     6661 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     6721 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     6781 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     6841 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     6901 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     6961 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaagcgacc
     7021 tgccgccaca aagaaggctg gacaggctaa gaagaagaaa gattacaaag acgatgacga
     7081 taagggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa
     7141 tcctggaccg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa gagcaacggc
     7201 tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag ctctctctag
     7261 cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac cttgtgcaga
     7321 actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc
     7381 gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac aggtgcttct
     7441 cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc cgacggcagt
     7501 tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaaacgc gttaagtcga
     7561 caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     7621 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     7681 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     7741 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     7801 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     7861 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     7921 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     7981 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     8041 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     8101 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta
     8161 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     8221 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct
     8281 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     8341 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     8401 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc
     8461 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     8521 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     8581 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     8641 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     8701 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     8761 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     8821 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     8881 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     8941 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     9001 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     9061 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     9121 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     9181 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     9241 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     9301 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     9361 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     9421 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     9481 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     9541 gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca tcggcatagt
     9601 atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg
     9661 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     9721 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     9781 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     9841 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
     9901 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
     9961 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
    10021 tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
    10081 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
    10141 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
    10201 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
    10261 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
    10321 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
    10381 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
    10441 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
    10501 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
    10561 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
    10621 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
    10681 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
    10741 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
    10801 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
    10861 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
    10921 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
    10981 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
    11041 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
    11101 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
    11161 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
    11221 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
    11281 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
    11341 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
    11401 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
    11461 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
    11521 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
    11581 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
    11641 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
    11701 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
    11761 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
    11821 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
    11881 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
    11941 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
    12001 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
    12061 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
    12121 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
    12181 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
    12241 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
    12301 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
    12361 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
    12421 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
//
