LOCUS       pLCV2-CDK12(hU6-       13280 bp DNA     circular SYN 31-AUG-2023
DEFINITION  Replaces original lentiCRISPRv1 (Addgene Plasmid 49535) and produces
            ~10-fold higher titer virus. 3rd generation lentiviral backbone..
ACCESSION   .
VERSION     .
KEYWORDS    pCLV2-CDK12(hU6-sg2-mU6-sg3)-Blast
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13280)
  AUTHORS   Sanjana NE, Shalem O, Zhang F
  TITLE     Improved vectors and genome-wide libraries for CRISPR screening.
  JOURNAL   Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047.
  PUBMED    25075903
REFERENCE   2  (bases 1 to 13280)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 31, 2023 from SnapGene 7.0.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nmeth"; 
            journalName: "Nat Methods"; date: "2014-08"; volume: "11"; issue: 
            "8"; pages: "783-4"
FEATURES             Location/Qualifiers
     source          1..13280
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..199
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     154..174
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
                     /note="color: black; sequence: CGCAAATGGGCGGTAGGCGTG"
     LTR             217..397
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
                     /note="color: #ffe4c4"
     misc_feature    444..569
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
                     /note="color: #ffe4c4"
     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."
                     /note="color: #ffe4c4"
     misc_feature    1822..1939
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
                     /note="color: #ffe4c4"
     promoter        1990..2230
                     /label=hU6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     1990..2010
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
                     /note="color: black; sequence: GAGGGCCTATTTCCCATGATT"
     primer_bind     2161..2180
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
                     /note="color: black; sequence: GACTATCATATGCTTACCGT"
     misc_feature    2240..2259
                     /label=CDK12(sg2)
                     /note="color: #ff9900"
     misc_RNA        2260..2335
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
                     /note="color: #00ccff; direction: RIGHT"
     misc_feature    2336..2341
                     /label=Terminator
                     /note="color: #800000"
     promoter        complement(2343..2361)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(2371..2387)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     promoter        2389..2702
                     /label=mU6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     2389..2407
                     /label=mU6-Fwd
                     /note="color: black; sequence: GATCCGACGCCGCCATCTC; added: 
                     2023-08-16"
     misc_feature    2705..2724
                     /label=CDK12(sg3)
                     /note="color: #ff0000"
     misc_feature    2725..2800
                     /label=gRNA scaffold
                     /note="color: #00ccff"
     misc_feature    2801..2806
                     /label=Terminator
                     /note="color: #800000"
     promoter        2862..3073
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     complement(3057..3073)
                     /label=EF1a-Rev
                     /note="color: black; sequence: ctgtgttctggcggcaa; added: 
                     2022-03-24"
     regulatory      3092..3101
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
     CDS             3098..7201
                     /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"
                     /note="color: #993366"
                     /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             7202..7249
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /note="color: #993366"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7250..7273
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /note="color: #cc99b2"
                     /translation="DYKDDDDK"
     CDS             7283..7339
                     /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."
                     /note="color: #cc99b2
                     Cleavage site after base 7336"
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7340..7738
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /note="color: #ccffcc"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    7754..8342
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /note="color: #ffffff"
     primer_bind     complement(7807..7827)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
                     /note="color: black; sequence: CATAGCGTAAAAGGAGCAACA"
     CDS             complement(8225..8236)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /note="color: #cc99b2
                     Cleavage site after base 8224"
                     /translation="IEGR"
     LTR             8414..8647
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
                     /note="color: #ffe4c4"
     primer_bind     complement(8673..8690)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
                     /note="color: black; sequence: TAGAAGGCACAGTCGAGG"
     polyA_signal    8679..8903
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     rep_origin      8949..9377
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     primer_bind     complement(9036..9055)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
                     /note="color: black; sequence: AGGGAAGAAAGCGAAAGGAG"
     primer_bind     9246..9267
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
                     /note="color: black; sequence: GTGGACTCTTGTTCCAAACTGG"
     primer_bind     complement(9386..9406)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
                     /note="color: black; sequence: ACCCTAACTGACACACATTCC"
     promoter        9391..9720
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
                     /note="color: #ffffff; direction: RIGHT"
     rep_origin      9571..9706
                     /label=SV40 ori
                     /note="SV40 origin of replication"
                     /note="color: #ffff00"
     primer_bind     9633..9652
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
                     /note="color: black; sequence: TATTTATGCAGAGGCCGAGG"
     promoter        9768..9815
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
                     /note="color: #ffffff; direction: RIGHT"
     CDS             9834..10208
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /note="color: #ccffcc"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    10338..10459
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(10375..10394)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
                     /note="color: black; sequence: GAAATTTGTGATGCTATTGC"
     primer_bind     10429..10448
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
                     /note="color: black; sequence: GTGGTTTGTCCAAACTCATC"
     primer_bind     complement(10508..10524)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     primer_bind     complement(10508..10524)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
                     /note="color: black; sequence: CAGGAAACAGCTATGAC"
     primer_bind     complement(10521..10543)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
                     /note="color: black; sequence: AGCGGATAACAATTTCACACAGG"
     protein_bind    10532..10548
                     /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)."
                     /note="color: #31849b"
     promoter        complement(10556..10586)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 10556 .. 10562 / #ffffff / -10
                        2: 10563 .. 10580 / #ffffff
                        3: 10581 .. 10586 / #ffffff / -35"
     protein_bind    10601..10622
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     primer_bind     complement(10739..10756)
                     /label=L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     rep_origin      complement(10910..11498)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     complement(10990..11009)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="color: black; sequence: GGGAAACGCCTGGTATCTTT"
     CDS             complement(11669..12529)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 11669 .. 12460 / #ccffcc
                        2: 12461 .. 12529 / #ccffcc / signal sequence
                     Cleavage site after base 12460"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     12292..12311
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
                     /note="color: black; sequence: ATAATACCGCGCCACATAGC"
     promoter        complement(12530..12634)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(12709..12728)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /note="color: black; sequence: CGGCATCAGAGCAGATTGTA"
     enhancer        12900..13279
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #ffffff"
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 gaaacaccgt ggccttcaaa ctagaccgag ttttagagct agaaatagca
     2281 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     2341 tccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaga tccgacgccg
     2401 ccatctctag gcccgcgccg gccccctcgc acagacttgt gggagaagct cggctactcc
     2461 cctgccccgg ttaatttgca tataatattt cctagtaact atagaggctt aatgtgcgat
     2521 aaaagacaga taatctgttc tttttaatac tagctacatt ttacatgata ggcttggatt
     2581 tctataagag atacaaatac taaattatta ttttaaaaaa cagcacaaaa ggaaactcac
     2641 cctaactgta aagtaattgt gtgttttgag actataaata tcccttggag aaaagccttg
     2701 tttggacaaa cagaaagcga ctgggtttta gagctagaaa tagcaagtta aaataaggct
     2761 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgaat tcgctagcta
     2821 ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc
     2881 cacagtcccc gagaagttgg ggggaggggt cggcaattga tccggtgcct agagaaggtg
     2941 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg
     3001 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc
     3061 cgccagaaca caggaccggt tctagagcgc tgccaccatg gacaagaagt acagcatcgg
     3121 cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
     3181 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
     3241 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
     3301 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
     3361 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
     3421 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
     3481 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
     3541 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
     3601 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
     3661 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
     3721 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
     3781 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
     3841 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     3901 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
     3961 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
     4021 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
     4081 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
     4141 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
     4201 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
     4261 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
     4321 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
     4381 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
     4441 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
     4501 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
     4561 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
     4621 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
     4681 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
     4741 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
     4801 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
     4861 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
     4921 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
     4981 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
     5041 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
     5101 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
     5161 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
     5221 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
     5281 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
     5341 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
     5401 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
     5461 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
     5521 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
     5581 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa
     5641 ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
     5701 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
     5761 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
     5821 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
     5881 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
     5941 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
     6001 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
     6061 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt
     6121 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
     6181 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
     6241 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga
     6301 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
     6361 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
     6421 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
     6481 taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa
     6541 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
     6601 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
     6661 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
     6721 cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct
     6781 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
     6841 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
     6901 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
     6961 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
     7021 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
     7081 caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat
     7141 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga
     7201 caagcgacct gccgccacaa agaaggctgg acaggctaag aagaagaaag attacaaaga
     7261 cgatgacgat aagggatccg gcgcaacaaa cttctctctg ctgaaacaag ccggagatgt
     7321 cgaagagaat cctggaccga tggccaagcc tttgtctcaa gaagaatcca ccctcattga
     7381 aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc
     7441 agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg
     7501 accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac
     7561 ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg
     7621 acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca
     7681 gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaaac
     7741 gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     7801 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     7861 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     7921 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     7981 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     8041 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     8101 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     8161 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     8221 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     8281 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     8341 gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     8401 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct
     8461 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     8521 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     8581 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     8641 tctagcaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     8701 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     8761 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     8821 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     8881 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     8941 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     9001 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     9061 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     9121 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     9181 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     9241 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     9301 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     9361 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     9421 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     9481 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     9541 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     9601 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     9661 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     9721 aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat
     9781 catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca
     9841 agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct
     9901 ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc
     9961 gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc
    10021 tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt
    10081 ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg
    10141 ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc
    10201 aggactgaca cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
    10261 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
    10321 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
    10381 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
    10441 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
    10501 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
    10561 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
    10621 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
    10681 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
    10741 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
    10801 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
    10861 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
    10921 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
    10981 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
    11041 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
    11101 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
    11161 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
    11221 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
    11281 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
    11341 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
    11401 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
    11461 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
    11521 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
    11581 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
    11641 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
    11701 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
    11761 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
    11821 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
    11881 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
    11941 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
    12001 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
    12061 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
    12121 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
    12181 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
    12241 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
    12301 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
    12361 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
    12421 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
    12481 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
    12541 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
    12601 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
    12661 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
    12721 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
    12781 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
    12841 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
    12901 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
    12961 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
    13021 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
    13081 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
    13141 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
    13201 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
    13261 agtcatcgct attaccatgg
//
