LOCUS       sequence-329709-       11704 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 11704)
  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 11704)
  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-gIfngr1-2
FEATURES             Location/Qualifiers
     source          1..11704
                     /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        1987..2062
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2110..2227
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2296..2507
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2527..2536
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2536..2559
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2566..2586
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2611..6711
                     /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             2611..6711
                     /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             6712..6759
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6775..6831
                     /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             6832..7545
                     /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    7561..8149
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7614..7634)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8032..8043)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8221..8454
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8532..8653
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8569..8588)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8623..8642
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      8693..8828
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(8848..8867)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8849..8867)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8877..8894)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8877..8893)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8886..8908)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9035..9490
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9122..9141)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9332..9353
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9516..9620
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9621..9689,9690..10481)
                     /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(9839..9858)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10652..11240
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11141..11160
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11394..11411
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    11528..11549
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(11564..11569,11570..11587,11588..11594)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11602..11618
                     /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     11607..11629
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     11626..11642
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11626..11642
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     11661..11681
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        11663..11681
                     /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 acaccgggta ttcccagcat
     1981 acgacagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     2041 aaaagtggca ccgagtcggt gcttttttga attctagatc ttgagacaaa tggcagtatt
     2101 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     2161 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     2221 aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc tcgagtggct
     2281 ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     2341 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     2401 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     2461 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg
     2521 ggatccgcca ccatggatta caaagacgat gacgataaga tggccccaaa gaagaagcgg
     2581 aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc
     2641 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
     2701 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg
     2761 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
     2821 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
     2881 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
     2941 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
     3001 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
     3061 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
     3121 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
     3181 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc
     3241 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
     3301 gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac
     3361 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
     3421 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
     3481 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     3541 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     3601 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     3661 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag
     3721 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     3781 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     3841 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     3901 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     3961 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     4021 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc
     4081 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     4141 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc
     4201 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg
     4261 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     4321 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     4381 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     4441 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     4501 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     4561 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg
     4621 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     4681 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc
     4741 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     4801 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     4861 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     4921 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     4981 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg
     5041 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     5101 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc
     5161 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc
     5221 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg
     5281 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     5341 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     5401 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg
     5461 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
     5521 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc
     5581 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc
     5641 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag
     5701 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
     5761 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag
     5821 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     5881 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     5941 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     6001 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     6061 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     6121 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     6181 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     6241 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     6301 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     6361 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     6421 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     6481 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     6541 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     6601 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     6661 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caagcgtcct
     6721 gctgctacta agaaagctgg tcaagctaag aaaaagaaag ctagcggcag cggcgccacc
     6781 aacttcagcc tgctgaagca ggccggcgac gtggaggaga accccggccc catggtgtct
     6841 aagggcgaag agctgattaa ggagaacatg cacatgaagc tgtatatgga gggcaccgtg
     6901 aacaaccacc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag
     6961 accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct
     7021 accagcttca tgtacggcag cagaaccttc atcaaccaca cccagggcat ccccgacttc
     7081 tttaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg
     7141 ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc
     7201 aagatcagag gggtgaactt cccatccaac ggccctgtga tgcagaagaa aacactcggc
     7261 tgggaggcca acaccgagat gctgtacccc gctgacggcg gcctggaagg cagaagcgac
     7321 atggccctga agctcgtggg cgggggccac ctgatctgca acttcaagac cacatacaga
     7381 tccaagaaac ccgctaagaa cctcaagatg cccggcgtct actatgtgga ccacagactg
     7441 gaaagaatca aggaggccga caaagagacc tacgtcgagc agcacgaggt ggctgtggcc
     7501 agatactgcg acctccctag caaactgggg cacaaactta attgaacgcg ttaagtcgac
     7561 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     7621 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     7681 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     7741 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     7801 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     7861 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     7921 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     7981 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     8041 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     8101 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa
     8161 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     8221 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
     8281 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     8341 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     8401 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt
     8461 atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag
     8521 agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     8581 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     8641 tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc
     8701 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     8761 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     8821 tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     8881 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     8941 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     9001 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     9061 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     9121 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     9181 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     9241 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     9301 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     9361 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     9421 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     9481 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     9541 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     9601 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     9661 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     9721 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     9781 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     9841 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     9901 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     9961 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
    10021 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
    10081 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
    10141 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
    10201 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
    10261 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
    10321 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
    10381 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
    10441 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
    10501 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
    10561 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
    10621 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
    10681 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
    10741 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
    10801 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
    10861 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
    10921 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
    10981 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
    11041 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
    11101 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
    11161 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
    11221 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
    11281 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
    11341 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
    11401 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
    11461 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
    11521 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
    11581 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
    11641 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc
    11701 aagc
//
