LOCUS       sequence-273058-       12824 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a human AMPKa1-targeting sgRNA and Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12824)
  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 12824)
  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-Blast-sgAMPKa1
FEATURES             Location/Qualifiers
     source          1..12824
                     /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        2260..2335
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2397..2608
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2627..2636
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2633..6736
                     /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             2633..6736
                     /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             6737..6784
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6785..6808
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6818..6874
                     /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             6875..7273
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    7298..7886
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7351..7371)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7769..7780)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7958..8191
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8217..8234)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8223..8447
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8493..8921
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8580..8599)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8790..8811
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8930..8950)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8935..9264
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9115..9250
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9177..9196
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9312..9359
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9378..9752
                     /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    9882..10003
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9919..9938)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9973..9992
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10052..10068)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10052..10068)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10065..10087)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10076..10092
                     /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(10100..10106,10107..10124,10125..10130))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10145..10166
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10283..10300)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10454..11042)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10534..10553)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11213..12004,12005..12073))
                     /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     11836..11855
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12074..12178)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12253..12272)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12444..12823
                     /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 gaaacaccgc tggtgtggat tattgtcacg ttttagagct agaaatagca
     2281 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     2341 tgaattcgct agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc
     2401 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg
     2461 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     2521 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     2581 tcgcaacggg tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa
     2641 gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga
     2701 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
     2761 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
     2821 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     2881 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     2941 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
     3001 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
     3061 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
     3121 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
     3181 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
     3241 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
     3301 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc
     3361 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
     3421 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
     3481 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
     3541 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
     3601 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
     3661 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
     3721 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
     3781 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
     3841 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
     3901 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
     3961 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
     4021 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
     4081 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
     4141 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga
     4201 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
     4261 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
     4321 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
     4381 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
     4441 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
     4501 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
     4561 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
     4621 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
     4681 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
     4741 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
     4801 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
     4861 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
     4921 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
     4981 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
     5041 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
     5101 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc
     5161 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa
     5221 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
     5281 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc
     5341 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
     5401 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
     5461 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
     5521 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
     5581 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
     5641 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
     5701 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
     5761 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
     5821 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt
     5881 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
     5941 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa
     6001 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
     6061 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
     6121 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
     6181 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
     6241 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
     6301 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
     6361 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
     6421 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
     6481 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
     6541 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
     6601 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
     6661 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
     6721 tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa
     6781 gaaagattac aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa
     6841 acaagccgga gatgtcgaag agaatcctgg accgatggcc aagcctttgt ctcaagaaga
     6901 atccaccctc attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta
     6961 cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata
     7021 tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc
     7081 agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc
     7141 ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa
     7201 ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt
     7261 gtgggagggc taagaattcg atatcaagct tatcggtaat caacctctgg attacaaaat
     7321 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     7381 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     7441 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     7501 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     7561 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     7621 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     7681 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     7741 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     7801 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     7861 gatctccctt tgggccgcct ccccgcgtcg actttaagac caatgactta caaggcagct
     7921 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     7981 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     8041 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     8101 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     8161 cccttttagt cagtgtggaa aatctctagc agggcccgtt taaacccgct gatcagcctc
     8221 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     8281 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     8341 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     8401 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     8461 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     8521 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     8581 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     8641 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     8701 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     8761 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     8821 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     8881 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     8941 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     9001 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     9061 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     9121 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     9181 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     9241 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     9301 gatcagcacg tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca
     9361 aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg
     9421 acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg
     9481 aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg
     9541 accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt
     9601 acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga
     9661 ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact
     9721 gcgtgcactt cgtggccgag gagcaggact gacacgtgct acgagatttc gattccaccg
     9781 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     9841 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     9901 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     9961 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
    10021 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
    10081 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
    10141 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
    10201 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
    10261 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
    10321 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
    10381 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
    10441 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
    10501 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
    10561 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
    10621 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
    10681 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
    10741 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
    10801 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
    10861 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
    10921 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
    10981 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
    11041 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
    11101 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
    11161 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
    11221 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
    11281 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
    11341 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
    11401 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
    11461 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
    11521 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
    11581 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
    11641 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
    11701 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
    11761 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
    11821 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
    11881 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
    11941 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
    12001 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
    12061 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
    12121 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
    12181 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
    12241 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
    12301 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
    12361 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
    12421 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
    12481 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
    12541 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
    12601 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
    12661 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
    12721 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
    12781 tacttggcag tacatctacg tattagtcat cgctattacc atgg
//
