LOCUS       sequence-273052-       13012 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a human BRSK1-targeting sgRNA and Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13012)
  AUTHORS   Hollstein PE, Eichner LJ, Brun SN, Kamireddy A, Svensson RU, Vera 
            LI, Ross DS, Rymoff TJ, Hutchins A, Galvez HM, Williams AE, 
            Shokhirev MN, Screaton RA, Berdeaux R, Shaw RJ
  TITLE     The AMPK-Related Kinases SIK1 and SIK3 Mediate Key Tumor-Suppressive
            Effects of LKB1 in NSCLC.
  JOURNAL   Cancer Discov. 2019 Nov;9(11):1606-1627. doi: 
            10.1158/2159-8290.CD-18-1261. Epub 2019 Jul 26.
  PUBMED    31350328
REFERENCE   2  (bases 1 to 13012)
  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"; doi: 
            "10.1158/2159-8290.CD-18-1261"; journalName: "Cancer Discov"; date: 
            "2019-11"; volume: "9"; issue: "11"; pages: "1606-1627"
COMMENT     Sequence Label: pLenti-Puro-sgBRSK1
FEATURES             Location/Qualifiers
     source          1..13012
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..199
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     154..174
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             217..397
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    444..569
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1062..1295
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1480..1524
                     /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    1822..1939
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1990..2238
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1990..2010
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2161..2180
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2259..2334
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2396..2607
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2626..2635
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2632..6735
                     /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             2632..6735
                     /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             6736..6783
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6784..6807
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6817..6873
                     /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             6874..7470
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     7367..7387
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7486..8074
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7539..7559)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7957..7968)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8146..8379
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8405..8422)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8411..8635
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8681..9109
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8768..8787)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8978..8999
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9118..9138)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9123..9452
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9303..9438
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9365..9384
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9500..9547
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9566..9940
                     /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    10070..10191
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10107..10126)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10161..10180
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10240..10256)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10240..10256)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10253..10275)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10264..10280
                     /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(10288..10294,10295..10312,10313..10318))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10333..10354
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10471..10488)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10642..11230)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10722..10741)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11401..12192,12193..12261))
                     /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     12024..12043
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12262..12366)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12441..12460)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12632..13011
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
       61 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      181 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
      241 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      301 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      361 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      421 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      481 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      541 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      601 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      661 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      721 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
      781 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
      841 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
      901 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
      961 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1021 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1081 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1141 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1201 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1261 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1321 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1381 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1441 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1501 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1561 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1621 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1681 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1741 agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca
     1801 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     1861 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     1921 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat
     1981 taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2041 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
     2101 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     2161 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     2221 tggaaaggac gaaacaccgg gtgagctatt cgactaccgt tttagagcta gaaatagcaa
     2281 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     2341 gaattcgcta gctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca
     2401 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgatccggt
     2461 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
     2521 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
     2581 cgcaacgggt ttgccgccag aacacaggac cggttctaga gcgctgccac catggacaag
     2641 aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac
     2701 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc
     2761 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg
     2821 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa
     2881 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag
     2941 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg
     3001 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg
     3061 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag
     3121 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag
     3181 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc
     3241 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa
     3301 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc
     3361 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa
     3421 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc
     3481 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc
     3541 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag
     3601 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg
     3661 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt
     3721 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg
     3781 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg
     3841 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg
     3901 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc
     3961 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc
     4021 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac
     4081 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc
     4141 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag
     4201 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag
     4261 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag
     4321 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg
     4381 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac
     4441 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg
     4501 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc
     4561 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc
     4621 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg
     4681 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc
     4741 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac
     4801 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg
     4861 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg
     4921 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg
     4981 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac
     5041 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac
     5101 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct
     5161 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac
     5221 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg
     5281 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc
     5341 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa
     5401 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac
     5461 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg
     5521 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac
     5581 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag
     5641 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc
     5701 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg
     5761 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc
     5821 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg
     5881 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc
     5941 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag
     6001 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg
     6061 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg
     6121 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa
     6181 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg
     6241 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga
     6301 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag
     6361 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag
     6421 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc
     6481 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga
     6541 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc
     6601 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg
     6661 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct
     6721 cagctgggag gcgacaagcg acctgccgcc acaaagaagg ctggacaggc taagaagaag
     6781 aaagattaca aagacgatga cgataaggga tccggcgcaa caaacttctc tctgctgaaa
     6841 caagccggag atgtcgaaga gaatcctgga ccgaccgagt acaagcccac ggtgcgcctc
     6901 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     6961 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     7021 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     7081 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     7141 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     7201 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc
     7261 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     7321 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     7381 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     7441 tggtgcatga cccgcaagcc cggtgcctga acgcgttaag tcgacaatca acctctggat
     7501 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     7561 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     7621 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     7681 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     7741 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     7801 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     7861 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     7921 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     7981 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     8041 acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca
     8101 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     8161 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
     8221 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     8281 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     8341 ccctcagacc cttttagtca gtgtggaaaa tctctagcag ggcccgttta aacccgctga
     8401 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     8461 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     8521 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     8581 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     8641 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     8701 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     8761 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     8821 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     8881 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     8941 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     9001 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     9061 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     9121 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     9181 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     9241 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     9301 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     9361 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     9421 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     9481 tcggatctga tcagcacgtg ttgacaatta atcatcggca tagtatatcg gcatagtata
     9541 atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc cggtgctcac
     9601 cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga
     9661 cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc
     9721 ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga
     9781 cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc
     9841 ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc
     9901 cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga
     9961 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
    10021 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
    10081 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
    10141 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
    10201 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
    10261 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
    10321 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
    10381 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
    10441 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
    10501 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
    10561 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
    10621 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
    10681 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
    10741 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
    10801 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
    10861 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
    10921 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
    10981 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
    11041 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
    11101 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
    11161 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
    11221 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
    11281 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
    11341 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
    11401 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
    11461 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
    11521 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
    11581 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
    11641 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
    11701 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
    11761 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
    11821 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
    11881 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
    11941 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
    12001 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
    12061 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
    12121 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
    12181 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
    12241 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
    12301 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
    12361 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
    12421 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
    12481 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
    12541 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
    12601 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
    12661 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
    12721 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
    12781 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
    12841 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
    12901 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
    12961 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat gg
//
