LOCUS       sequence-321916-       11590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral vector expressing Cas9 and an sgRNA targeting HK2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11590)
  AUTHORS   Rossiter NJ, Huggler KS, Adelmann CH, Keys HR, Soens RW, Sabatini 
            DM, Cantor JR
  TITLE     CRISPR screens in physiologic medium reveal conditionally essential 
            genes in human cells.
  JOURNAL   Cell Metab. 2021 Feb 23. pii: S1550-4131(21)00061-9. doi: 
            10.1016/j.cmet.2021.02.005.
  PUBMED    33651980
REFERENCE   2  (bases 1 to 11590)
  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: "Cell Metab.
            2021 Feb 23. pii: S1550-4131(21)00061-9. doi: 
            10.1016/j.cmet.2021.02.005."
COMMENT     Sequence Label: pLentiCRISPR-v1-sgHK2_2
FEATURES             Location/Qualifiers
     source          1..11590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     134..154
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        136..154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        182..408
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             409..589
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    636..761
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1254..1487
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1672..1716
                     /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"
     promoter        1894..2142
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1894..1914
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2065..2084
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2164..2239
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2287..2404
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2473..2684
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2704..2713
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2713..2736
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2743..2763
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2788..6888
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2788..6888
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6889..6936
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6952..7008
                     /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             7009..7608
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(7009..7028)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7505..7525
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7624..8212
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7677..7697)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8095..8106)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8284..8517
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8595..8716
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8632..8651)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8686..8705
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8756..8891
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8911..8930)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8912..8930)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8940..8957)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8940..8956)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8949..8971)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9098..9553
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9185..9204)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9395..9416
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9579..9683
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9684..9752,9753..10544)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(9902..9921)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10715..11303
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11204..11223
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11457..11474
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta
      181 atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat
      241 gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat
      301 cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg
      361 ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg
      421 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      481 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      541 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
      601 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
      661 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
      721 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
      781 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
      841 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
      901 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
      961 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1021 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1081 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1141 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1201 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1261 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1321 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1381 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1441 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1501 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1561 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     1621 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     1681 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     1741 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     1801 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     1861 cagacccacc tcccaacccc gaggggaccc agagagggcc tatttcccat gattccttca
     1921 tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac
     1981 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     2041 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     2101 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgtaagcgg ttccgcaagg
     2161 agagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     2221 agtggcaccg agtcggtgct tttttgaatt ctagatcttg agacaaatgg cagtattcat
     2281 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     2341 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     2401 ttttcgggtt tattacaggg acagcagaga tccactttgg cgccggctcg agtggctccg
     2461 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
     2521 tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
     2581 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
     2641 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgacgcggga
     2701 tccgccacca tggattacaa agacgatgac gataagatgg ccccaaagaa gaagcggaag
     2761 gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct ggacatcggc
     2821 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc
     2881 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg
     2941 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac
     3001 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag
     3061 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag
     3121 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac
     3181 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg
     3241 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc
     3301 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac
     3361 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg
     3421 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag
     3481 aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc
     3541 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac
     3601 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc
     3661 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc
     3721 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg
     3781 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc
     3841 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc
     3901 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag
     3961 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac
     4021 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc
     4081 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg
     4141 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc
     4201 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc
     4261 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
     4321 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag
     4381 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg
     4441 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
     4501 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
     4561 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
     4621 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
     4681 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
     4741 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
     4801 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
     4861 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
     4921 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
     4981 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
     5041 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
     5101 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
     5161 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
     5221 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
     5281 ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc
     5341 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc
     5401 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt
     5461 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
     5521 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
     5581 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa
     5641 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
     5701 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
     5761 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
     5821 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
     5881 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
     5941 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
     6001 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
     6061 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
     6121 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
     6181 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
     6241 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc
     6301 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
     6361 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
     6421 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
     6481 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
     6541 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
     6601 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
     6661 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
     6721 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
     6781 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
     6841 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgtcctgct
     6901 gctactaaga aagctggtca agctaagaaa aagaaagcta gcggcagcgg cgccaccaac
     6961 ttcagcctgc tgaagcaggc cggcgacgtg gaggagaacc ccggccccat gaccgagtac
     7021 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     7081 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     7141 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     7201 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     7261 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     7321 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     7381 ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     7441 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     7501 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     7561 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc
     7621 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     7681 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     7741 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     7801 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     7861 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     7921 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     7981 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     8041 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     8101 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     8161 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt
     8221 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     8281 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     8341 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     8401 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     8461 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     8521 cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
     8581 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     8641 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     8701 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc
     8761 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     8821 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     8881 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     8941 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     9001 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     9061 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
     9121 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     9181 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     9241 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     9301 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     9361 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     9421 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     9481 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     9541 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     9601 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     9661 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     9721 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     9781 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     9841 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     9901 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     9961 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
    10021 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
    10081 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
    10141 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
    10201 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
    10261 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
    10321 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
    10381 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
    10441 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
    10501 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
    10561 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
    10621 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
    10681 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
    10741 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
    10801 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
    10861 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
    10921 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
    10981 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
    11041 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
    11101 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
    11161 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
    11221 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
    11281 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
    11341 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
    11401 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
    11461 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
    11521 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
    11581 gcaacgcaat
//
