LOCUS       sequence-249673-        9290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR gRNA against human TSC2 with Cas9 from S. pyogenes and 
            2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9290)
  AUTHORS   Walton ZE, Patel CH, Brooks RC, Yu Y, Ibrahim-Hashim A, Riddle M, 
            Porcu A, Jiang T, Ecker BL, Tameire F, Koumenis C, Weeraratna AT, 
            Welsh DK, Gillies R, Alwine JC, Zhang L, Powell JD, Dang CV
  TITLE     Acid Suspends the Circadian Clock in Hypoxia through Inhibition of 
            mTOR.
  JOURNAL   Cell. 2018 Jun 28;174(1):72-87.e32. doi: 10.1016/j.cell.2018.05.009.
            Epub 2018 May 31.
  PUBMED    29861175
REFERENCE   2  (bases 1 to 9290)
  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: 
            "10.1016/j.cell.2018.05.009"; journalName: "Cell"; date: 
            "2018-06-28- 28"; volume: "174"; issue: "1"; pages: "72-87.e32"
COMMENT     Sequence Label: PX458-sgTSC2
FEATURES             Location/Qualifiers
     source          1..9290
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        651..899
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     651..671
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     822..841
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        921..996
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1093..1378
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1380..1656
                     /label=chicken beta-actin promoter
     intron          1657..1884
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1896..1905
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1905..1970
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1977..1997
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2022..6122
                     /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             2022..6122
                     /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             6123..6170
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6186..6239
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(6240..6242,6243..6953)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6240..6242,6243..6953)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6543..6562)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6890..6911
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6981..6998)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6987..7194
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7203..7343
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7203..7332
                     /label=AAV2 ITR
     rep_origin      7418..7873
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7505..7524)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7715..7736
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7890..7909)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8009..8031
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8069..8087)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8155..8259
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8260..8328,8329..9120)
                     /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(8478..8497)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat
      661 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
      721 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      781 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      841 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
      901 tctgctgaag gccatcgtgc gttttagagc tagaaatagc aagttaaaat aaggctagtc
      961 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga gctagaaata
     1021 gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca gacaaatggc
     1081 tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     1141 cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt
     1201 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     1261 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac
     1321 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     1381 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     1441 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg
     1501 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
     1561 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
     1621 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
     1681 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
     1741 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag
     1801 aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac
     1861 ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggactat aaggaccacg
     1921 acggagacta caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa
     1981 agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg
     2041 gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc
     2101 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga
     2161 tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg
     2221 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca
     2281 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     2341 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     2401 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     2461 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     2521 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     2581 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     2641 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     2701 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     2761 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     2821 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     2881 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     2941 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     3001 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     3061 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     3121 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     3181 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     3241 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     3301 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     3361 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     3421 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     3481 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     3541 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     3601 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     3661 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     3721 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     3781 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     3841 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     3901 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     3961 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     4021 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     4081 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     4141 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     4201 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     4261 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     4321 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     4381 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     4441 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     4501 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga
     4561 aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     4621 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     4681 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     4741 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     4801 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     4861 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     4921 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     4981 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
     5041 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     5101 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     5161 ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg
     5221 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     5281 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
     5341 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
     5401 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
     5461 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca
     5521 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
     5581 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
     5641 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
     5701 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
     5761 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg
     5821 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
     5881 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
     5941 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
     6001 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
     6061 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
     6121 acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag gaattcggca
     6181 gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccag
     6241 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     6301 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     6361 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     6421 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     6481 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     6541 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     6601 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     6661 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     6721 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     6781 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     6841 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     6901 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aaggaattct
     6961 aactagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     7021 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     7081 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     7141 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc
     7201 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     7261 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     7321 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     7381 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     7441 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     7501 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     7561 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     7621 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     7681 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     7741 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     7801 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     7861 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     7921 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     7981 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     8041 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     8101 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     8161 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     8221 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     8281 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     8341 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     8401 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     8461 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     8521 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     8581 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     8641 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     8701 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     8761 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     8821 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     8881 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     8941 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     9001 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     9061 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     9121 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     9181 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     9241 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
