LOCUS       sequence-197213-       13014 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Introducing a gRNA targeting human TTF-1 (NKX2-1) into cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13014)
  AUTHORS   Lai SC, Phelps CA, Short AM, Dutta SM, Mu D
  TITLE     Thyroid transcription factor 1 enhances cellular statin sensitivity 
            via perturbing cholesterol metabolism.
  JOURNAL   Oncogene. 2018 Mar 19. pii: 10.1038/s41388-018-0174-7. doi: 
            10.1038/s41388-018-0174-7.
  PUBMED    29551766
REFERENCE   2  (bases 1 to 13014)
  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: "Oncogene. 
            2018 Mar 19. pii: 10.1038/s41388-018-0174-7. doi: 
            10.1038/s41388-018-0174-7."
COMMENT     Sequence Label: lentiCRISPRv2-TTF-1-gRNA
FEATURES             Location/Qualifiers
     source          1..13014
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..181
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    228..353
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    846..1079
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1264..1308
                     /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    1606..1723
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1774..2022
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1774..1794
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1945..1964
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2045..2120
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2182..2393
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2412..2421
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2418..6521
                     /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             2418..6521
                     /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             6522..6569
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6570..6593
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6603..6659
                     /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             6660..7256
                     /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     7153..7173
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7272..7860
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7325..7345)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7743..7754)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7932..8165
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8191..8208)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8197..8421
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8467..8895
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8554..8573)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8764..8785
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8904..8924)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8909..9238
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9089..9224
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9151..9170
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9286..9333
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9352..9726
                     /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    9856..9977
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9893..9912)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9947..9966
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10026..10042)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10026..10042)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10039..10061)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10050..10066
                     /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(10074..10080,10081..10098,10099..10104))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10119..10140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10257..10274)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10428..11016)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10508..10527)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11187..11978,11979..12047))
                     /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     11810..11829
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12048..12152)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12227..12246)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12418..12797
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        12799..12997
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     12952..12972
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     1501 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
     1561 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
     1621 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     1681 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     1741 cagcagagat ccagtttggt taattaaggt accgagggcc tatttcccat gattccttca
     1801 tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac
     1861 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     1921 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     1981 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgggggctc cgctggcggc
     2041 gtacgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     2101 aagtggcacc gagtcggtgc ttttttgaat tcgctagcta ggtcttgaaa ggagtgggaa
     2161 ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
     2221 ggggaggggt cggcaattga tccggtgcct agagaaggtg gcgcggggta aactgggaaa
     2281 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
     2341 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggaccggt
     2401 tctagagcgc tgccaccatg gacaagaagt acagcatcgg cctggacatc ggcaccaact
     2461 ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc
     2521 tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca
     2581 gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac
     2641 ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg
     2701 acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc
     2761 ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca
     2821 tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct
     2881 atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga
     2941 accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc
     3001 tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca
     3061 gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga
     3121 atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca
     3181 acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc
     3241 tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga
     3301 acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg
     3361 cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc
     3421 tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga
     3481 gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt
     3541 tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca
     3601 gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc
     3661 acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg
     3721 acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc
     3781 tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc
     3841 cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga
     3901 tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt
     3961 acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga
     4021 gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga
     4081 ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct
     4141 tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc
     4201 acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca
     4261 ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac
     4321 ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga
     4381 gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt
     4441 ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc
     4501 agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg
     4561 gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga
     4621 agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca
     4681 agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga
     4741 agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga
     4801 tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact
     4861 acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg
     4921 actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca
     4981 aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg
     5041 tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga
     5101 gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg
     5161 gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc
     5221 tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag
     5281 tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag
     5341 tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa
     5401 ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg
     5461 tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca
     5521 agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg
     5581 gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg
     5641 ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg
     5701 tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga
     5761 acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg
     5821 acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca
     5881 agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg
     5941 agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga
     6001 tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg
     6061 cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact
     6121 tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga
     6181 aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg
     6241 agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca
     6301 acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc
     6361 tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga
     6421 ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc
     6481 tgtacgagac acggatcgac ctgtctcagc tgggaggcga caagcgacct gccgccacaa
     6541 agaaggctgg acaggctaag aagaagaaag attacaaaga cgatgacgat aagggatccg
     6601 gcgcaacaaa cttctctctg ctgaaacaag ccggagatgt cgaagagaat cctggaccga
     6661 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     6721 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     6781 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     6841 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     6901 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     6961 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     7021 cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg
     7081 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     7141 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     7201 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc
     7261 gttaagtcga caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     7321 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     7381 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     7441 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     7501 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     7561 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     7621 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     7681 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     7741 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     7801 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     7861 gtcgacttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     7921 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg
     7981 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     8041 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     8101 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     8161 tagcagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     8221 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     8281 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     8341 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     8401 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     8461 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     8521 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     8581 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     8641 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     8701 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     8761 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     8821 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     8881 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     8941 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     9001 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     9061 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     9121 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     9181 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     9241 gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca
     9301 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag
     9361 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg
     9421 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg
     9481 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg
     9541 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc
     9601 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
     9661 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag
     9721 gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     9781 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     9841 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     9901 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     9961 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
    10021 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
    10081 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
    10141 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
    10201 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
    10261 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
    10321 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
    10381 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
    10441 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
    10501 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
    10561 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
    10621 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
    10681 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
    10741 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
    10801 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
    10861 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
    10921 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
    10981 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
    11041 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
    11101 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
    11161 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
    11221 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
    11281 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
    11341 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
    11401 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
    11461 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
    11521 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
    11581 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
    11641 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
    11701 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
    11761 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
    11821 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
    11881 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
    11941 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
    12001 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
    12061 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
    12121 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
    12181 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
    12241 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
    12301 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
    12361 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
    12421 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
    12481 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
    12541 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
    12601 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
    12661 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
    12721 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
    12781 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
    12841 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
    12901 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
    12961 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tact
//
