LOCUS       sequence-362603-       11690 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets ABL2 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11690)
  AUTHORS   Hu M, Lei XY, Larson JD, McAlonis M, Ford K, McDonald D, Mach K, 
            Rusert JM, Wechsler-Reya RJ, Mali P
  TITLE     Integrated genome and tissue engineering enables screening of cancer
            vulnerabilities in physiologically relevant perfusable ex vivo 
            cultures.
  JOURNAL   Biomaterials. 2022 Jan;280:121276. doi: 
            10.1016/j.biomaterials.2021.121276. Epub 2021 Dec 2.
  PUBMED    34890975
REFERENCE   2  (bases 1 to 11690)
  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: 
            "Biomaterials."; date: "2022-01"; volume: "280:121276. doi"; pages: 
            " 10.1016/j.biomaterials.2021.121276. Epub 2021 Dec 2"
COMMENT     Sequence Label: CROP_C9_Puro_ABL2
FEATURES             Location/Qualifiers
     source          1..11690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..30
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     14..30
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     49..69
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        51..69
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        97..323
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             324..504
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    551..676
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1169..1402
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1587..1631
                     /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    1812..1929
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2118..2329
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2348..2357
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2354..6457
                     /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             2354..6457
                     /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             6458..6505
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6506..6529
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6539..6595
                     /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             6596..7195
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6596..6615)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7092..7112
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7211..7799
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7264..7284)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7682..7693)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7955..8202
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7955..7975
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8126..8145
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8224..8299
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8352..8532
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8610..8731
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8647..8666)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8701..8720
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8771..8906
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8926..8945)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8927..8945)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8955..8972)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8955..8971)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8964..8986)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9113..9568
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9200..9219)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9410..9431
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9594..9698
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9699..9767,9768..10559)
                     /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(9917..9936)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10730..11318
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11219..11238
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11472..11489
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11606..11627
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11642..11647,11648..11665,11666..11672)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(11680..11690,1..6)
                     /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     join(11685..11690,1..17)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct
       61 cactaaaggg aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt
      121 agtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc
      181 gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga
      241 cgggtctgac atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta
      301 agtgcctagc tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc
      361 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
      421 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
      481 agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa
      541 accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga
      601 ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga
      661 tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg
      721 gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga
      781 gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat
      841 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
      901 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
      961 tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc
     1021 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
     1081 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
     1141 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
     1201 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
     1261 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
     1321 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
     1381 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
     1441 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
     1501 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
     1561 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat
     1621 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
     1681 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga
     1741 atagagttag gcagggatat tcaccattat cgtttcagaa tcttgagaca aatggcagta
     1801 ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata
     1861 gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt
     1921 caaaattttc gggtttatta cagggacagc agagatccac tttggcgccg gctcgagggg
     1981 gcccgggtgc aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc
     2041 ttctaggtct tgaaaggagt gggaattggc tccgtaggtc ttgaaaggag tgggaattgg
     2101 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
     2161 aggggtcggc aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
     2221 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
     2281 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg accggttcta
     2341 gagcgctgcc accatggaca agaagtacag catcggcctg gacatcggca ccaactctgt
     2401 gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg
     2461 caacaccgac cggcacagca tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg
     2521 cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa
     2581 gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag
     2641 cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca
     2701 ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta
     2761 ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct
     2821 ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc
     2881 cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt
     2941 cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact
     3001 gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg
     3061 cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt
     3121 cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga
     3181 caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct
     3241 gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc
     3301 cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa
     3361 agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa
     3421 gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat
     3481 caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga
     3541 ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct
     3601 gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa
     3661 ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc
     3721 caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg
     3781 gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac
     3841 caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga
     3901 gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa
     3961 gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa
     4021 ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga
     4081 ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga
     4141 tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct
     4201 ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct
     4261 gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata
     4321 caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg
     4381 caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct
     4441 gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca
     4501 gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg
     4561 catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc
     4621 cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa
     4681 cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct
     4741 gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct
     4801 gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta
     4861 cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt
     4921 gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt
     4981 gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa
     5041 gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt
     5101 catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga
     5161 ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat
     5221 caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg
     5281 cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc
     5341 cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta
     5401 cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta
     5461 cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga
     5521 gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa
     5581 gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa
     5641 aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag
     5701 cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag
     5761 ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa
     5821 actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa
     5881 gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat
     5941 caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc
     6001 tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct
     6061 gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca
     6121 gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt
     6181 ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa
     6241 gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac
     6301 caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta
     6361 caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta
     6421 cgagacacgg atcgacctgt ctcagctggg aggcgacaag cgacctgccg ccacaaagaa
     6481 ggctggacag gctaagaaga agaaagatta caaagacgat gacgataagg gatccggcgc
     6541 aacaaacttc tctctgctga aacaagccgg agatgtcgaa gagaatcctg gaccgatgac
     6601 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     6661 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     6721 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     6781 caaggtgtgg gtcgcggacg acggcgccgc cgtggcggtc tggaccacgc cggagagcgt
     6841 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     6901 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     6961 gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     7021 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     7081 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     7141 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg
     7201 ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     7261 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     7321 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     7381 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     7441 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     7501 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     7561 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     7621 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     7681 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     7741 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg
     7801 tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     7861 aaggggggac tggaagggct aattcactcc caacgaagac aagatttcac cattatcgtt
     7921 tcagacccac ctcccaaccc cgaggggacc cagagagggc ctatttccca tgattccttc
     7981 atatttgcat atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa
     8041 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     8101 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     8161 cgatttcttg gctttatata tcttgtggaa agacgaaaca ccgctgctgc cccggatccc
     8221 gcggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     8281 agtggcaccg agtcggtgct tttttaagct tggcgtaact agatcttgag acactgcttt
     8341 ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     8401 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     8461 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     8521 aaatctctag cagtacgtat agtagttcat gtcatcttat tattcagtat ttataacttg
     8581 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca
     8641 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     8701 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct
     8761 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     8821 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     8881 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg
     8941 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     9001 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     9061 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc
     9121 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     9181 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     9241 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     9301 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     9361 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     9421 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     9481 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     9541 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     9601 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     9661 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     9721 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     9781 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     9841 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     9901 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     9961 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
    10021 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
    10081 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
    10141 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
    10201 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
    10261 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
    10321 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
    10381 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
    10441 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
    10501 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
    10561 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
    10621 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
    10681 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
    10741 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
    10801 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
    10861 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
    10921 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
    10981 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
    11041 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
    11101 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
    11161 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
    11221 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
    11281 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
    11341 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
    11401 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
    11461 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
    11521 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
    11581 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
    11641 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
//
