LOCUS       sequence-396598-       13013 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing Cas9 and sgRNA targeting human RB1 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13013)
  AUTHORS   Wang ME, Chen J, Lu Y, Bawcom AR, Wu J, Ou J, Asara JM, Armstrong 
            AJ, Wang Q, Li L, Wang Y, Huang J, Chen M
  TITLE     RB1-deficient prostate tumor growth and metastasis are vulnerable to
            ferroptosis induction via the E2F/ACSL4 axis.
  JOURNAL   J Clin Invest. 2023 Mar 16:e166647. doi: 10.1172/JCI166647.
  PUBMED    36928314
REFERENCE   2  (bases 1 to 13013)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Clin 
            Invest. 2023 Mar 16:e166647. doi: 10.1172/JCI166647."
COMMENT     Sequence Label: LentiCRISPR v2 RB1 sgRNA #2
FEATURES             Location/Qualifiers
     source          1..13013
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..199
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     154..174
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             217..397
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    444..569
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1062..1295
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1480..1524
                     /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"
     misc_feature    1822..1939
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1990..2238
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1990..2010
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2161..2180
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2260..2335
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2397..2608
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2627..2636
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2633..6736
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2633..6736
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6737..6784
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6785..6808
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6818..6874
                     /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             6875..7471
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     7368..7388
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    7487..8075
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7540..7560)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7958..7969)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8147..8380
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8406..8423)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8412..8636
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8682..9110
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8769..8788)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8979..9000
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9119..9139)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9124..9453
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9304..9439
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9366..9385
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9501..9548
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9567..9941
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    10071..10192
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10108..10127)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10162..10181
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10241..10257)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10241..10257)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10254..10276)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10265..10281
                     /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)."
     promoter        complement(join(10289..10295,10296..10313,10314..10319))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10334..10355
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10472..10489)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10643..11231)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10723..10742)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11402..12193,12194..12262))
                     /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     12025..12044
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12263..12367)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12442..12461)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12633..13012
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
       61 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      181 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
      241 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      301 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      361 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      421 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      481 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      541 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      601 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      661 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      721 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
      781 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
      841 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
      901 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt
      961 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca
     1021 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg
     1081 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc
     1141 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg
     1201 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg
     1261 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa
     1321 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag
     1381 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta
     1441 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg
     1501 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat
     1561 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta
     1621 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc
     1681 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac
     1741 agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca
     1801 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     1861 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     1921 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat
     1981 taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2041 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
     2101 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     2161 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     2221 tggaaaggac gaaacaccgg gttctttgag caacatgggg ttttagagct agaaatagca
     2281 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     2341 tgaattcgct agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc
     2401 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg
     2461 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
     2521 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
     2581 tcgcaacggg tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa
     2641 gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga
     2701 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat
     2761 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg
     2821 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca
     2881 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga
     2941 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt
     3001 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt
     3061 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa
     3121 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa
     3181 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc
     3241 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga
     3301 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc
     3361 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa
     3421 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg
     3481 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag
     3541 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa
     3601 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct
     3661 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat
     3721 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat
     3781 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg
     3841 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct
     3901 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat
     3961 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc
     4021 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga
     4081 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc
     4141 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga
     4201 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga
     4261 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca
     4321 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt
     4381 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga
     4441 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct
     4501 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt
     4561 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag
     4621 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct
     4681 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac
     4741 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca
     4801 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt
     4861 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat
     4921 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg
     4981 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa
     5041 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta
     5101 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc
     5161 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa
     5221 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg
     5281 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc
     5341 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga
     5401 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta
     5461 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt
     5521 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca
     5581 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa
     5641 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc
     5701 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat
     5761 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat
     5821 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt
     5881 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg
     5941 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa
     6001 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt
     6061 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct
     6121 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga
     6181 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct
     6241 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg
     6301 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga
     6361 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa
     6421 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc
     6481 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag
     6541 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc
     6601 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct
     6661 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc
     6721 tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa
     6781 gaaagattac aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa
     6841 acaagccgga gatgtcgaag agaatcctgg accgaccgag tacaagccca cggtgcgcct
     6901 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     6961 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     7021 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     7081 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     7141 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     7201 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt
     7261 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     7321 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     7381 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     7441 ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga
     7501 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     7561 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     7621 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     7681 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     7741 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     7801 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     7861 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     7921 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     7981 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     8041 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac
     8101 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     8161 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
     8221 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     8281 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     8341 tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg
     8401 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     8461 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     8521 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     8581 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     8641 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     8701 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     8761 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     8821 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     8881 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     8941 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     9001 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     9061 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     9121 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     9181 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     9241 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     9301 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     9361 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     9421 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     9481 ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat
     9541 aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca
     9601 ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg
     9661 acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg
     9721 cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg
     9781 acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc
     9841 cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg
     9901 ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg
     9961 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
    10021 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
    10081 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
    10141 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
    10201 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
    10261 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
    10321 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
    10381 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
    10441 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
    10501 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
    10561 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
    10621 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
    10681 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
    10741 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
    10801 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
    10861 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
    10921 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
    10981 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
    11041 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
    11101 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
    11161 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
    11221 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
    11281 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
    11341 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
    11401 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
    11461 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
    11521 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
    11581 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
    11641 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
    11701 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
    11761 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
    11821 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
    11881 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
    11941 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
    12001 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
    12061 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
    12121 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
    12181 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
    12241 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
    12301 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
    12361 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
    12421 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
    12481 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
    12541 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
    12601 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata
    12661 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
    12721 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
    12781 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
    12841 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
    12901 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
    12961 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tgg
//
