LOCUS       sequence-321918-       11589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral vector expressing Cas9 and an sgRNA targeting GLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11589)
  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 11589)
  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-sgGLS_2
FEATURES             Location/Qualifiers
     source          1..11589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    96..329
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             514..558
                     /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        736..984
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     736..756
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     907..926
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1005..1080
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    1128..1245
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1314..1525
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1545..1554
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1554..1577
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1584..1604
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1629..5729
                     /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             1629..5729
                     /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             5730..5777
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5793..5849
                     /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             5850..6449
                     /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(5850..5869)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6346..6366
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    6465..7053
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6518..6538)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6936..6947)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7125..7358
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7436..7557
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7473..7492)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7527..7546
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7597..7732
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7752..7771)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7753..7771)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7781..7798)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7781..7797)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7790..7812)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7939..8394
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8026..8045)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8236..8257
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        8420..8524
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8525..8593,8594..9385)
                     /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(8743..8762)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9556..10144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10045..10064
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10298..10315
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    10432..10453
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(10468..10473,10474..10491,10492..10498)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10506..10522
                     /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     10511..10533
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     10530..10546
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10530..10546
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     10565..10585
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        10567..10585
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        10613..10839
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             10840..11020
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11067..11192
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
ORIGIN
        1 tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga
       61 agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg
      121 ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa
      181 ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag
      241 catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg
      301 gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt
      361 tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg
      421 aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac
      481 tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta
      541 gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa
      601 ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct
      661 atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc
      721 ccgaggggac ccagagaggg cctatttccc atgattcctt catatttgca tatacgatac
      781 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa
      841 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
      901 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
      961 atcttgtgga aaggacgaaa caccgagcac gcatccgcag cccggtttta gagctagaaa
     1021 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     1081 ttttttgaat tctagatctt gagacaaatg gcagtattca tccacaattt taaaagaaaa
     1141 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     1201 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     1261 gacagcagag atccactttg gcgccggctc gagtggctcc ggtgcccgtc agtgggcaga
     1321 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc
     1381 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
     1441 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     1501 caacgggttt gccgccagaa cacaggtgtc gtgacgcggg atccgccacc atggattaca
     1561 aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc
     1621 cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
     1681 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
     1741 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
     1801 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
     1861 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
     1921 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
     1981 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
     2041 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
     2101 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     2161 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     2221 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     2281 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     2341 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     2401 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     2461 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     2521 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     2581 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     2641 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     2701 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     2761 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     2821 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     2881 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     2941 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     3001 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     3061 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     3121 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     3181 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     3241 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     3301 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     3361 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     3421 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     3481 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     3541 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     3601 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     3661 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     3721 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     3781 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     3841 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     3901 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     3961 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     4021 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     4081 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     4141 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     4201 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     4261 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     4321 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     4381 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     4441 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     4501 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     4561 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     4621 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     4681 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     4741 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     4801 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     4861 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     4921 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     4981 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     5041 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     5101 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     5161 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     5221 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     5281 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     5341 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     5401 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     5461 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     5521 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     5581 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     5641 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     5701 ggatcgacct gtctcagctg ggaggcgaca agcgtcctgc tgctactaag aaagctggtc
     5761 aagctaagaa aaagaaagct agcggcagcg gcgccaccaa cttcagcctg ctgaagcagg
     5821 ccggcgacgt ggaggagaac cccggcccca tgaccgagta caagcccacg gtgcgcctcg
     5881 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
     5941 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
     6001 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     6061 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     6121 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     6181 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggagtct
     6241 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     6301 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     6361 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     6421 ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa cctctggatt
     6481 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     6541 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     6601 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     6661 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     6721 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     6781 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     6841 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     6901 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     6961 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     7021 cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa
     7081 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     7141 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
     7201 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     7261 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     7321 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca
     7381 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt
     7441 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     7501 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     7561 tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     7621 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     7681 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg
     7741 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     7801 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     7861 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     7921 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     7981 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     8041 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     8101 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     8161 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     8221 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     8281 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     8341 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     8401 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     8461 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     8521 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     8581 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     8641 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     8701 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     8761 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     8821 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     8881 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     8941 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     9001 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     9061 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     9121 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     9181 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     9241 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     9301 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     9361 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     9421 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     9481 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     9541 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     9601 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     9661 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     9721 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     9781 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     9841 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     9901 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     9961 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
    10021 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
    10081 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
    10141 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
    10201 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
    10261 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
    10321 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
    10381 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
    10441 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
    10501 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
    10561 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt aatgtagtct
    10621 tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca tgccttacaa
    10681 ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga tcgtgcctta
    10741 ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt gccgcattgc
    10801 agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg gttagaccag
    10861 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
    10921 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
    10981 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
    11041 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
    11101 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
    11161 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
    11221 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
    11281 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
    11341 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
    11401 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
    11461 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
    11521 cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
    11581 agtgaatta
//
