LOCUS       sequence-319221-       13013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  knock out SLC7A11/xCT in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13013)
  AUTHORS   Liu X, Olszewski K, Zhang Y, Lim EW, Shi J, Zhang X, Zhang J, Lee H,
            Koppula P, Lei G, Zhuang L, You MJ, Fang B, Li W, Metallo CM, 
            Poyurovsky MV, Gan B
  TITLE     Cystine transporter regulation of pentose phosphate pathway 
            dependency and disulfide stress exposes a targetable metabolic 
            vulnerability in cancer.
  JOURNAL   Nat Cell Biol. 2020 Apr;22(4):476-486. doi: 
            10.1038/s41556-020-0496-x. Epub 2020 Mar 30.
  PUBMED    32231310
REFERENCE   2  (bases 1 to 13013)
  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: 
            "10.1038/s41556-020-0496-x"; journalName: "Nat Cell Biol"; date: 
            "2020-04"; volume: "22"; issue: "4"; pages: "476-486"
COMMENT     Sequence Label: lentiCRISPR v2-sgSLC7A11/xCT-2
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(79..99)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        84..413
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      264..399
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     326..345
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        461..508
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             527..901
                     /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    1031..1152
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1068..1087)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1122..1141
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1201..1217)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1201..1217)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1214..1236)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1225..1241
                     /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(1249..1255,1256..1273,1274..1279))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1294..1315
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1432..1449)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1603..2191)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1683..1702)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2362..3153,3154..3222))
                     /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     2985..3004
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3223..3327)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3402..3421)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3593..3972
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3974..4172
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4127..4147
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4190..4370
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4417..4542
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5035..5268
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5453..5497
                     /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    5795..5912
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5963..6211
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5963..5983
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6134..6153
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        6233..6308
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        6370..6581
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      6600..6609
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6606..10709
                     /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             6606..10709
                     /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             10710..10757
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             10758..10781
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             10791..10847
                     /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             10848..11444
                     /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     11341..11361
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    11460..12048
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(11513..11533)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(11931..11942)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             12120..12353
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(12379..12396)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    12385..12609
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(12655..13013,1..70)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12742..12761)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12952..12973
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
       61 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
      121 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
      181 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
      241 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
      301 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
      361 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
      421 cgggagcttg tatatccatt ttcggatctg atcagcacgt gttgacaatt aatcatcggc
      481 atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac
      541 cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga
      601 ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga
      661 cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg
      721 ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa
      781 cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga
      841 gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg
      901 acacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
      961 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     1021 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     1081 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     1141 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     1201 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     1261 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     1321 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     1381 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     1441 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     1501 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1561 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1621 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1681 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1741 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1801 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1861 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1921 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     1981 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2041 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2101 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2161 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2221 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2281 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2341 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2401 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2461 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     2521 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2581 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2641 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     2701 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     2761 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     2821 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     2881 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     2941 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     3001 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     3061 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3121 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3181 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     3241 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3301 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     3361 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     3421 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     3481 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     3541 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga
     3601 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     3661 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     3721 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     3781 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     3841 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     3901 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     3961 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     4021 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     4081 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     4141 aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt gcctgtactg ggtctctctg
     4201 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     4261 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     4321 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
     4381 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
     4441 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg
     4501 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
     4561 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta
     4621 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
     4681 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
     4741 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
     4801 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt
     4861 aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
     4921 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
     4981 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
     5041 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
     5101 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
     5161 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
     5221 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
     5281 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
     5341 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
     5401 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     5461 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     5521 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     5581 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     5641 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     5701 tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac tgcgtgcgcc
     5761 aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg
     5821 ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat
     5881 tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc
     5941 cagtttggtt aattaaggta ccgagggcct atttcccatg attccttcat atttgcatat
     6001 acgatacaag gctgttagag agataattag aattaatttg actgtaaaca caaagatatt
     6061 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
     6121 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
     6181 tttatatatc ttgtggaaag gacgaaacac cgaagtatta cgcggttgcc acgttttaga
     6241 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     6301 gtcggtgctt ttttgaattc gctagctagg tcttgaaagg agtgggaatt ggctccggtg
     6361 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
     6421 gcaattgatc cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
     6481 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
     6541 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggaccggttc tagagcgctg
     6601 ccaccatgga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
     6661 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
     6721 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
     6781 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
     6841 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
     6901 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     6961 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     7021 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     7081 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     7141 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     7201 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     7261 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     7321 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     7381 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     7441 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     7501 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     7561 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     7621 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     7681 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     7741 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     7801 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     7861 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     7921 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     7981 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     8041 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     8101 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     8161 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     8221 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     8281 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     8341 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     8401 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     8461 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     8521 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     8581 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     8641 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     8701 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     8761 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     8821 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     8881 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     8941 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     9001 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     9061 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     9121 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     9181 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     9241 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     9301 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     9361 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     9421 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     9481 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     9541 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     9601 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     9661 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     9721 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     9781 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     9841 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     9901 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     9961 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
    10021 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
    10081 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
    10141 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
    10201 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
    10261 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
    10321 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
    10381 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
    10441 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
    10501 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
    10561 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
    10621 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
    10681 ggatcgacct gtctcagctg ggaggcgaca agcgacctgc cgccacaaag aaggctggac
    10741 aggctaagaa gaagaaagat tacaaagacg atgacgataa gggatccggc gcaacaaact
    10801 tctctctgct gaaacaagcc ggagatgtcg aagagaatcc tggaccgacc gagtacaagc
    10861 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
    10921 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
    10981 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
    11041 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
    11101 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
    11161 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
    11221 ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
    11281 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
    11341 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
    11401 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaacgcgt taagtcgaca
    11461 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
    11521 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
    11581 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
    11641 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
    11701 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
    11761 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
    11821 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
    11881 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
    11941 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
    12001 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcgt cgactttaag
    12061 accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact
    12121 ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct
    12181 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
    12241 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
    12301 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagggcccg
    12361 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
    12421 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
    12481 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
    12541 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
    12601 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
    12661 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
    12721 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
    12781 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
    12841 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
    12901 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
    12961 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat tta
//
