LOCUS       sequence-330049-       11705 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses spCas9, tRFP and a gRNA targeting mouse Ifngr1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11705)
  AUTHORS   Yu M, Peng Z, Qin M, Liu Y, Wang J, Zhang C, Lin J, Dong T, Wang L, 
            Li S, Yang Y, Xu S, Guo W, Zhang X, Shi M, Peng H, Luo X, Zhang H, 
            Zhang L, Li Y, Yang XP, Sun S
  TITLE     Interferon-gamma induces tumor resistance to anti-PD-1 immunotherapy
            by promoting YAP phase separation.
  JOURNAL   Mol Cell. 2021 Feb 13. pii: S1097-2765(21)00010-1. doi: 
            10.1016/j.molcel.2021.01.010.
  PUBMED    33606996
REFERENCE   2  (bases 1 to 11705)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell. 
            2021 Feb 13. pii: S1097-2765(21)00010-1. doi: 
            10.1016/j.molcel.2021.01.010."
COMMENT     Sequence Label: PL-CRISPR.EFS.tRFP-gIfngr1-1
FEATURES             Location/Qualifiers
     source          1..11705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /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        1717..1965
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1988..2063
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2111..2228
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2297..2508
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2528..2537
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2537..2560
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2567..2587
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2612..6712
                     /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             2612..6712
                     /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             6713..6760
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6776..6832
                     /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             6833..7546
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     misc_feature    7562..8150
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7615..7635)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8033..8044)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8222..8455
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8533..8654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8570..8589)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8624..8643
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8694..8829
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8849..8868)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8850..8868)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8878..8895)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8878..8894)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8887..8909)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9036..9491
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9123..9142)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9333..9354
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9517..9621
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9622..9690,9691..10482)
                     /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(9840..9859)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10653..11241
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11142..11161
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11395..11412
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11529..11550
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11565..11570,11571..11588,11589..11595)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11603..11619
                     /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     11608..11630
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11627..11643
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11627..11643
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     11662..11682
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        11664..11682
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgactt gaaccctgtc
     1981 gtatgctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
     2041 aaaaagtggc accgagtcgg tgcttttttg aattctagat cttgagacaa atggcagtat
     2101 tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag
     2161 tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc
     2221 aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg ctcgagtggc
     2281 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     2341 ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     2401 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     2461 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc
     2521 gggatccgcc accatggatt acaaagacga tgacgataag atggccccaa agaagaagcg
     2581 gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat
     2641 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     2701 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
     2761 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     2821 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     2881 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     2941 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     3001 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     3061 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     3121 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     3181 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     3241 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     3301 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     3361 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     3421 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     3481 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     3541 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     3601 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     3661 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     3721 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     3781 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     3841 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     3901 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     3961 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     4021 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     4081 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     4141 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     4201 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     4261 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     4321 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     4381 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     4441 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     4501 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     4561 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     4621 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     4681 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     4741 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     4801 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     4861 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     4921 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     4981 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     5041 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     5101 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc
     5161 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     5221 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     5281 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     5341 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     5401 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     5461 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     5521 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     5581 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     5641 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     5701 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     5761 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     5821 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     5881 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     5941 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     6001 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     6061 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     6121 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     6181 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     6241 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     6301 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     6361 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     6421 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     6481 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     6541 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     6601 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     6661 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaagcgtcc
     6721 tgctgctact aagaaagctg gtcaagctaa gaaaaagaaa gctagcggca gcggcgccac
     6781 caacttcagc ctgctgaagc aggccggcga cgtggaggag aaccccggcc ccatggtgtc
     6841 taagggcgaa gagctgatta aggagaacat gcacatgaag ctgtatatgg agggcaccgt
     6901 gaacaaccac cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca
     6961 gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc
     7021 taccagcttc atgtacggca gcagaacctt catcaaccac acccagggca tccccgactt
     7081 ctttaagcag tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg
     7141 gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt
     7201 caagatcaga ggggtgaact tcccatccaa cggccctgtg atgcagaaga aaacactcgg
     7261 ctgggaggcc aacaccgaga tgctgtaccc cgctgacggc ggcctggaag gcagaagcga
     7321 catggccctg aagctcgtgg gcgggggcca cctgatctgc aacttcaaga ccacatacag
     7381 atccaagaaa cccgctaaga acctcaagat gcccggcgtc tactatgtgg accacagact
     7441 ggaaagaatc aaggaggccg acaaagagac ctacgtcgag cagcacgagg tggctgtggc
     7501 cagatactgc gacctcccta gcaaactggg gcacaaactt aattgaacgc gttaagtcga
     7561 caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     7621 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     7681 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     7741 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     7801 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     7861 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     7921 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     7981 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     8041 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     8101 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta
     8161 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     8221 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct
     8281 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     8341 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     8401 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg
     8461 tatagtagtt catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca
     8521 gagagtgaga ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     8581 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     8641 atcaatgtat cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc
     8701 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     8761 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     8821 ttggaggcct agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     8881 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     8941 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     9001 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag
     9061 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     9121 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     9181 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     9241 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     9301 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     9361 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     9421 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     9481 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     9541 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     9601 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     9661 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     9721 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     9781 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     9841 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     9901 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     9961 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
    10021 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
    10081 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
    10141 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
    10201 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
    10261 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
    10321 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
    10381 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
    10441 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
    10501 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
    10561 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
    10621 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
    10681 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
    10741 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
    10801 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
    10861 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
    10921 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
    10981 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
    11041 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
    11101 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
    11161 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
    11221 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
    11281 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
    11341 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
    11401 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
    11461 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
    11521 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
    11581 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
    11641 gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg
    11701 caagc
//
