LOCUS       sequence-362610-       11691 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-One CRISPRko system with a guide RNA that targets CDK4 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_CDK4
FEATURES             Location/Qualifiers
     source          1..11691
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    5..21
                     /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     10..32
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     29..45
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     29..45
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     64..84
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        66..84
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        112..338
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             339..519
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    566..691
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1184..1417
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1602..1646
                     /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    1827..1944
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2133..2344
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2363..2372
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2369..6472
                     /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             2369..6472
                     /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             6473..6520
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6521..6544
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6554..6610
                     /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             6611..7210
                     /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(6611..6630)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7107..7127
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7226..7814
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7279..7299)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7697..7708)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        7970..8218
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7970..7990
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8141..8160
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8240..8315
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     LTR             8368..8548
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     polyA_signal    8626..8747
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8663..8682)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8717..8736
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8787..8922
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8942..8961)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8943..8961)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8971..8988)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8971..8987)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8980..9002)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9129..9584
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9216..9235)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9426..9447
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9610..9714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9715..9783,9784..10575)
                     /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(9933..9952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10746..11334
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11235..11254
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11488..11505
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11622..11643
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11658..11663,11664..11681,11682..11688)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
       61 cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt
      121 atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag
      181 gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat
      241 taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca
      301 gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga
      361 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
      421 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
      481 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
      541 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
      601 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
      661 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
      721 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
      781 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
      841 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
      901 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
      961 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     1021 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     1081 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     1141 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     1201 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     1261 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     1321 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     1381 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     1441 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     1501 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     1561 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     1621 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     1681 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     1741 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagaatcttg
     1801 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     1861 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     1921 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg
     1981 cgccggctcg agggggcccg ggtgcaaaga tggataaagt tttaaacaga gaggaatctt
     2041 tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgt aggtcttgaa
     2101 aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     2161 cgagaagttg gggggagggg tcggcaattg atccggtgcc tagagaaggt ggcgcggggt
     2221 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
     2281 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
     2341 acaggaccgg ttctagagcg ctgccaccat ggacaagaag tacagcatcg gcctggacat
     2401 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     2461 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
     2521 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     2581 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     2641 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     2701 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     2761 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     2821 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     2881 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     2941 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     3001 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     3061 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     3121 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     3181 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     3241 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     3301 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     3361 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     3421 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     3481 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     3541 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     3601 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     3661 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     3721 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     3781 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     3841 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     3901 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     3961 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     4021 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     4081 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     4141 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     4201 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     4261 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     4321 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     4381 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     4441 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     4501 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     4561 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     4621 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     4681 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     4741 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     4801 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     4861 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc
     4921 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     4981 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     5041 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     5101 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     5161 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     5221 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     5281 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     5341 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     5401 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     5461 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     5521 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     5581 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     5641 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     5701 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     5761 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     5821 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     5881 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     5941 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     6001 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     6061 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     6121 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     6181 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     6241 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     6301 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     6361 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     6421 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaagcgacc
     6481 tgccgccaca aagaaggctg gacaggctaa gaagaagaaa gattacaaag acgatgacga
     6541 taagggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa
     6601 tcctggaccg atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     6661 cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     6721 cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     6781 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgccgtgg cggtctggac
     6841 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     6901 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     6961 gcccaaggag cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa
     7021 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     7081 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     7141 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     7201 cggtgcctga acgcgttaag tcgacaatca acctctggat tacaaaattt gtgaaagatt
     7261 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     7321 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     7381 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     7441 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     7501 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     7561 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     7621 atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc
     7681 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     7741 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     7801 ggccgcctcc ccgcgtcgac tttaagacca atgacttaca aggcagctgt agatcttagc
     7861 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     7921 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccagag agggcctatt
     7981 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
     8041 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     8101 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     8161 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg
     8221 tctacatgct caaacaccag ttttagagct agaaatagca agttaaaata aggctagtcc
     8281 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt taagcttggc gtaactagat
     8341 cttgagacac tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg
     8401 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     8461 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     8521 ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca tcttattatt
     8581 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca
     8641 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     8701 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc
     8761 tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     8821 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     8881 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc
     8941 gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg
     9001 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     9061 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     9121 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     9181 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     9241 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     9301 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     9361 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     9421 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     9481 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     9541 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     9601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     9661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     9721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     9781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     9841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     9901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     9961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
    10021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
    10081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
    10141 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
    10201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
    10261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
    10321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
    10381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
    10441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
    10501 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
    10561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
    10621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
    10681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
    10741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
    10801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
    10861 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
    10921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
    10981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
    11041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
    11101 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
    11161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
    11221 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
    11281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
    11341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
    11401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
    11461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
    11521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
    11581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
    11641 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg t
//
