LOCUS       sequence-194444-       13735 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Lentiviral CRISPR-Cas9 delivery for SpCas9 and sgRNA. Coexpresses 
            eGFP via P2A cleavage site. SFFV Promoter driven.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13735)
  AUTHORS   Heckl D, Kowalczyk MS, Yudovich D, Belizaire R, Puram RV, McConkey 
            ME, Thielke A, Aster JC, Regev A, Ebert BL
  TITLE     Generation of mouse models of myeloid malignancy with combinatorial 
            genetic lesions using CRISPR-Cas9 genome editing.
  JOURNAL   Nat Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951.
  PUBMED    24952903
REFERENCE   2  (bases 1 to 13735)
  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: "Nat 
            Biotechnol. 2014 Jun 22. doi: 10.1038/nbt.2951."
COMMENT     Sequence Label: pL-CRISPR.SFFV.GFP
FEATURES             Location/Qualifiers
     source          1..13735
                     /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        3847..3922
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    3970..4087
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4145..4552
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      4552..4561
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4561..4584
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4591..4611
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4636..8736
                     /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             4636..8736
                     /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             8737..8784
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             8800..8856
                     /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             join(8857..8859,8860..8862,8863..9573)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(9163..9182)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9510..9531
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    9592..10180
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9645..9665)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10063..10074)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             10252..10485
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    10563..10684
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10600..10619)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10654..10673
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      10724..10859
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(10879..10898)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(10880..10898)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(10908..10925)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(10908..10924)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10917..10939)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      11066..11521
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11153..11172)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11363..11384
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        11547..11651
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11652..11720,11721..12512)
                     /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(11870..11889)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12683..13271
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     13172..13191
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     13425..13442
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    13559..13580
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(13595..13600,13601..13618,13619..13625)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13633..13649
                     /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     13638..13660
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     13657..13673
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     13657..13673
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     13692..13712
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        13694..13712
                     /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 acaccggaga cggttgtaaa
     1981 tgagcacaca aaatacacat gctaaaatat tatattctat gacctttata aaatcaacca
     2041 aaatcttctt tttaataact ttagtatcaa taattagaat ttttatgttc ctttttgcaa
     2101 acttttaata aaaatgagca aaataaaaaa acgctagttt tagtaactcg cgttgttttc
     2161 ttcaccttta ataatagcta ctccaccact tgttcctaag cggtcagctc ctgcttcaat
     2221 cattttttga gcatcttcaa atgttctaac tccaccagct gctttaacta aagcattgtc
     2281 tttaacaact gacttcatta gtttaacatc ttcaaatgtt gcacctgatt ttgaaaatcc
     2341 tgttgatgtt ttaacaaatt ctaatccagc ttcaacagct atttcacaag ctttcatgat
     2401 ttcttctttt gttaataaac aattttccat aatacattta acaacatgtg atccagctgc
     2461 tttttttaca gctttcatgt cttctaaaac taattcataa tttttgtctt ttaatgcacc
     2521 aatatttaat accatatcaa tttctgttgc accatcttta attgcttcag aaacttcgaa
     2581 tgcttttgta gctgttgtgc atgcacctag aggaaaacct acaacatttg ttattcctac
     2641 atttgtgcct tttaataatt ctttacaata gcttgttcaa tatgaattaa cacaaactgt
     2701 tgcaaaatca aattcaattg cttcatcaca taattgttta atttcagctt tcgtagcatc
     2761 ttgttttaat aatgtgtgat ctatatattt gtttagtttc attttttctc ctatatattc
     2821 atttttaatt ttaattcttt aataatttcg tctactttaa ctttagcgtt ttgaacagat
     2881 tcaccaacac ctataaaata aatttttagt ttaggttcag ttccacttgg gcgaacagca
     2941 aatcatgact tatcttctaa ataaaatttt agtaagtctt gtcctggcat attatacatt
     3001 ccatcgatgt agtcttcaac attaacaact ttaagtccag caatttgagt taagggtgtt
     3061 gctctcaatg atttcattaa tggttcaatt tttaatttct tttcttctgg tttaaaattc
     3121 aagtttaaag tgaaagtgta atatgcaccc atttctttaa ataaatcttc taaatagtct
     3181 actaatgttt tattttgttt tttataaaat caagcagcct ctgctattaa tatagaagct
     3241 tgtattccat ctttatctct agctgagtca tcaattacat atccataact ttcttcataa
     3301 gcaaaaacaa aatttaatcc gttatcttct tctttagcaa tttctctacc cattcattta
     3361 aatccagtta aagtttttac aatattaact ccatattttt catgagcgat tctatcaccc
     3421 aaatcacttg ttacaaaact tgaatataga gccggatttt ttggaatgct atttaagcgt
     3481 tttagatttg ataattttca atcaattaaa attggtcctg tttgatttcc atctaatctt
     3541 acaaaatgac catcatgttt tattgccatt ccaaatctgt cagcatctgg gtcattcata
     3601 ataataatat ctgcatcatg tttaatacca tattcaagcg gtatttttca tgcaggatca
     3661 aattctggat ttggatttac aacattttta aatgtttcat cttcaaatgc atgctcttca
     3721 acctcaataa cgttatatcc tgattcacgt aatatttttg gggtaaattt agttcctgtt
     3781 ccattaactg cgctaaaaat aatttttaaa tcttttttag cttcttgctc ttttttgtac
     3841 gtctctgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     3901 aaaagtggca ccgagtcggt gcttttttga attctagatc ttgagacaaa tggcagtatt
     3961 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     4021 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     4081 aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc tcgagcgagc
     4141 tgcagtaacg ccattttgca aggcatggaa aaataccaaa ccaagaatag agaagttcag
     4201 atcaagggcg ggtacatgaa aatagctaac gttgggccaa acaggatatc tgcggtgagc
     4261 agtttcggcc ccggcccggg gccaagaaca gatggtcacc gcagtttcgg ccccggcccg
     4321 aggccaagaa cagatggtcc ccagatatgg cccaaccctc agcagtttct taagacccat
     4381 cagatgtttc caggctcccc caaggacctg aaatgaccct gcgccttatt tgaattaacc
     4441 aatcagcctg cttctcgctt ctgttcgcgc gcttctgctt cccgagctct ataaaagagc
     4501 tcacaacccc tcactcggcg cgccagtcct ccgacagact gagtcggatc cgccaccatg
     4561 gattacaaag acgatgacga taagatggcc ccaaagaaga agcggaaggt cggtatccac
     4621 ggagtcccag cagccgacaa gaagtacagc atcggcctgg acatcggcac caactctgtg
     4681 ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc
     4741 aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc
     4801 gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag
     4861 aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc
     4921 ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac
     4981 cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac
     5041 cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg
     5101 gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc
     5161 gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc
     5221 gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg
     5281 agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc
     5341 ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc
     5401 gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac
     5461 aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg
     5521 tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc
     5581 ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa
     5641 gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag
     5701 aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc
     5761 aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag
     5821 gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg
     5881 ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac
     5941 cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc
     6001 aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg
     6061 aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc
     6121 aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag
     6181 tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag
     6241 cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac
     6301 cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac
     6361 tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat
     6421 ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg
     6481 gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg
     6541 aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac
     6601 accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc
     6661 aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg
     6721 atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag
     6781 ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc
     6841 atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc
     6901 gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac
     6961 agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg
     7021 aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg
     7081 cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac
     7141 gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg
     7201 ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg
     7261 aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag
     7321 ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc
     7381 atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac
     7441 tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc
     7501 accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc
     7561 gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc
     7621 ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac
     7681 gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac
     7741 ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag
     7801 atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag
     7861 ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa
     7921 aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc
     7981 gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc
     8041 cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa
     8101 ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag
     8161 aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc
     8221 aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct
     8281 gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg
     8341 tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag
     8401 ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc
     8461 tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag
     8521 caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc
     8581 aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac
     8641 accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac
     8701 gagacacgga tcgacctgtc tcagctggga ggcgacaagc gtcctgctgc tactaagaaa
     8761 gctggtcaag ctaagaaaaa gaaagctagc ggcagcggcg ccaccaactt cagcctgctg
     8821 aagcaggccg gcgacgtgga ggagaacccc ggccccatgg tgtctaaggg cgaagagctg
     8881 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     8941 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     9001 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     9061 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     9121 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     9181 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     9241 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     9301 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     9361 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     9421 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     9481 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     9541 gggatcactc tcggcatgga cgagctgtac aagtaaacgc gttaagtcga caatcaacct
     9601 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     9661 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     9721 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     9781 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     9841 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     9901 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     9961 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
    10021 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
    10081 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
    10141 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
    10201 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
    10261 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
    10321 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
    10381 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
    10441 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt
    10501 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
    10561 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
    10621 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
    10681 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc
    10741 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
    10801 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
    10861 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
    10921 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
    10981 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
    11041 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
    11101 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
    11161 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
    11221 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
    11281 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
    11341 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
    11401 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
    11461 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
    11521 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
    11581 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
    11641 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
    11701 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
    11761 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
    11821 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
    11881 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
    11941 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
    12001 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
    12061 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
    12121 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
    12181 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
    12241 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
    12301 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
    12361 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
    12421 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
    12481 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
    12541 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
    12601 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
    12661 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
    12721 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
    12781 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
    12841 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
    12901 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
    12961 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
    13021 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
    13081 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
    13141 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
    13201 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
    13261 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
    13321 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
    13381 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
    13441 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
    13501 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
    13561 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
    13621 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
    13681 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagc
//
