LOCUS       sequence-362658-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets PIGF gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11691)
  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 11691)
  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_PIGF
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..42
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        24..42
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        70..296
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             297..477
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    524..649
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1142..1375
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1560..1604
                     /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    1785..1902
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2091..2302
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2321..2330
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2327..6430
                     /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             2327..6430
                     /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             6431..6478
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6479..6502
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6512..6568
                     /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             6569..7168
                     /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(6569..6588)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7065..7085
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7184..7772
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7237..7257)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7655..7666)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7928..8176
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7928..7948
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8099..8118
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8198..8273
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8326..8506
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8584..8705
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8621..8640)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8675..8694
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8745..8880
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8900..8919)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8901..8919)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8929..8946)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8929..8945)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8938..8960)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9087..9542
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9174..9193)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9384..9405
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9568..9672
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9673..9741,9742..10533)
                     /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(9891..9910)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10704..11292
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11193..11212
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11446..11463
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11580..11601
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11616..11621,11622..11639,11640..11646)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11654..11670
                     /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     11659..11681
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(11678..11691,1..3)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(11678..11691,1..3)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg
       61 caagcttaat gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt
      121 agcaacatgc cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt
      181 ggtacgatcg tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca
      241 ctgaattgcc gcattgcaga gatattgtat ttaagtgcct agctcgatac ataaacgggt
      301 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
      361 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
      421 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg
      481 gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac
      541 tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa
      601 aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg
      661 ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata
      721 taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg
      781 cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac catcccttca
      841 gacaggatca gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca
      901 tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa
      961 caaaagtaag accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata
     1021 tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag
     1081 gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa
     1141 taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa
     1201 tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt
     1261 tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc
     1321 agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga
     1381 tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga
     1441 gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa
     1501 ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa
     1561 agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca
     1621 taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt
     1681 taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat
     1741 tatcgtttca gaatcttgag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     1801 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     1861 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     1921 agcagagatc cactttggcg ccggctcgag ggggcccggg tgcaaagatg gataaagttt
     1981 taaacagaga ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt
     2041 ggctccgtag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt gggcagagcg
     2101 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat ccggtgccta
     2161 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
     2221 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
     2281 cgggtttgcc gccagaacac aggaccggtt ctagagcgct gccaccatgg acaagaagta
     2341 cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
     2401 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
     2461 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa
     2521 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
     2581 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
     2641 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
     2701 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
     2761 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
     2821 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
     2881 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
     2941 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
     3001 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag
     3061 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
     3121 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
     3181 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
     3241 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     3301 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     3361 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
     3421 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     3481 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     3541 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     3601 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     3661 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     3721 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     3781 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
     3841 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
     3901 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     3961 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     4021 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     4081 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     4141 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     4201 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     4261 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     4321 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     4381 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
     4441 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     4501 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     4561 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     4621 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     4681 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     4741 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     4801 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag
     4861 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     4921 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     4981 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
     5041 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     5101 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     5161 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     5221 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     5281 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
     5341 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     5401 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     5461 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
     5521 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
     5581 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     5641 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
     5701 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
     5761 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     5821 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     5881 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     5941 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     6001 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     6061 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     6121 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     6181 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     6241 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     6301 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
     6361 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     6421 gggaggcgac aagcgacctg ccgccacaaa gaaggctgga caggctaaga agaagaaaga
     6481 ttacaaagac gatgacgata agggatccgg cgcaacaaac ttctctctgc tgaaacaagc
     6541 cggagatgtc gaagagaatc ctggaccgat gaccgagtac aagcccacgg tgcgcctcgc
     6601 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     6661 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     6721 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     6781 cgccgtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     6841 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     6901 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggagtctc
     6961 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     7021 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     7081 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     7141 gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac ctctggatta
     7201 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     7261 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     7321 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     7381 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     7441 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     7501 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     7561 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     7621 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     7681 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     7741 gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag
     7801 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     7861 tcccaacgaa gacaagattt caccattatc gtttcagacc cacctcccaa ccccgagggg
     7921 acccagagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     7981 tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     8041 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     8101 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     8161 gaaaggacga aacaccgaag ttctccaaga agagtgagtt ttagagctag aaatagcaag
     8221 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttta
     8281 agcttggcgt aactagatct tgagacactg ctttttgctt gtactgggtc tctctggtta
     8341 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     8401 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     8461 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtac gtatagtagt
     8521 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag
     8581 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     8641 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     8701 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc
     8761 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     8821 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     8881 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     8941 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     9001 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     9061 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     9121 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     9181 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     9241 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     9301 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     9361 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     9421 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     9481 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     9541 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     9601 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     9661 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     9721 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     9781 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     9841 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     9901 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     9961 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
    10021 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
    10081 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
    10141 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
    10201 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
    10261 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
    10321 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
    10381 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
    10441 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
    10501 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
    10561 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
    10621 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
    10681 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
    10741 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
    10801 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
    10861 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
    10921 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
    10981 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
    11041 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
    11101 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
    11161 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
    11221 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
    11281 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
    11341 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
    11401 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
    11461 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
    11521 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
    11581 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
    11641 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta t
//
