LOCUS       sequence-305764-       13500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lenti sgRNA cloning backbone with MS2 loops at tetraloop and 
            stemloop 2, dCas9-VP64, and blast resistance marker. Contains BsmBI 
            sites for insertion of spacer sequences..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13500)
  AUTHORS   Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham
            MD, Sanjana NE, Zhang F
  TITLE     Genome-scale CRISPR-Cas9 knockout and transcriptional activation 
            screening.
  JOURNAL   Nat Protoc. 2017 Apr;12(4):828-863. doi: 10.1038/nprot.2017.016. 
            Epub 2017 Mar 23.
  PUBMED    28333914
REFERENCE   2  (bases 1 to 13500)
  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"; doi: 
            "10.1038/nprot.2017.016"; journalName: "Nat Protoc"; date: 
            "2017-04"; volume: "12"; issue: "4"; pages: "828-863"
COMMENT     Sequence Label: lentiSAMv2
FEATURES             Location/Qualifiers
     source          1..13500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     33..52
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     286..303
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    420..441
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(456..461,462..479,480..486)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    494..510
                     /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     499..521
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     518..534
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     518..534
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    583..704
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(594..613)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     648..667
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(834..1208)
                     /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"
     promoter        complement(1227..1274)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(1322..1651)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1336..1471
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1390..1409)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     1636..1656
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1665..2093)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1775..1796)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1987..2006
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2139..2363
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2352..2369
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     LTR             2395..2575
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     3080..3096
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3081..3097
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    3100..3688
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3206..3217
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     3615..3635
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3713..4111)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(4112..4165)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(4187..4336)
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             complement(4361..4381)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(4436..5950,5951..5953,5954..8509,8510..8512,
                     8513..8536))
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(8537..8584)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     promoter        complement(8601..8812)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_RNA        complement(8899..8917)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        complement(8969..8987)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        complement(9025..9273)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(9083..9102)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(9253..9273)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    9323..9440
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(9738..9782)
                     /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    9967..10200
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    10693..10818
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             10865..11045
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(11063..11261)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(11088..11108)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        11263..11642
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     11814..11833
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        11908..12012
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(12013..12081,12082..12873)
                     /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(12231..12250)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(13044..13500,1..132)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
       61 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      121 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      181 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
      241 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
      301 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
      361 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
      421 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
      481 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
      541 tacgccaagc tctagctaga ggtcgacggt atacagacat gataagatac attgatgagt
      601 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
      661 ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg ggcgaagaac
      721 tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa aacgattccg
      781 aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtagcac gtgtcagtcc
      841 tgctcctcgg ccacgaagtg cacgcagttg ccggccgggt cgcgcagggc gaactcccgc
      901 ccccacggct gctcgccgat ctcggtcatg gccggcccgg aggcgtcccg gaagttcgtg
      961 gacacgacct ccgaccactc ggcgtacagc tcgtccaggc cgcgcaccca cacccaggcc
     1021 agggtgttgt ccggcaccac ctggtcctgg accgcgctga tgaacagggt cacgtcgtcc
     1081 cggaccacac cggcgaagtc gtcctccacg aagtcccggg agaacccgag ccggtcggtc
     1141 cagaactcga ccgctccggc gacgtcgcgc gcggtgagca ccggaacggc actggtcaac
     1201 ttggccatgg tttagttcct caccttgtcg tattatacta tgccgatata ctatgccgat
     1261 gattaattgt caacacgtgc tgatcagatc cgaaaatgga tatacaagct cccgggagct
     1321 ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg
     1381 cagaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat
     1441 gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg actatggttg
     1501 ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc
     1561 cacacctggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc
     1621 ctggggactt tccacaccct aactgacaca cattccacag aattaattcg cgttaaattt
     1681 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     1741 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     1801 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     1861 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     1921 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     1981 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     2041 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtggggata
     2101 ccccctagag ccccagctgg ttctttccgc ctcagaagcc atagagccca ccgcatcccc
     2161 agcatgcctg ctattgtctt cccaatcctc ccccttgctg tcctgcccca ccccaccccc
     2221 cagaatagaa tgacacctac tcagacaatg cgatgcaatt tcctcatttt attaggaaag
     2281 gacagtggga gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga
     2341 tggctggcaa ctagaaggca cagtcgaggc tgatcagcgg gtttaaacgg gccctgctag
     2401 agattttcca cactgactaa aagggtctga gggatctcta gttaccagag tcacacaaca
     2461 gacgggcaca cactacttga agcactcaag gcaagcttta ttgaggctta agcagtgggt
     2521 tccctagtta gccagagagc tcccaggctc agatctggtc taaccagaga gacccagtac
     2581 agtccggatg cagctctcgg gccatgtgat gaaatgctag gcggctgtca aacctccact
     2641 ctaatacttc tctctccggg tcatccatcc catgcaggct cacagggtgt aacaagcggg
     2701 tgttctctcc ttcattggct tcttctacct tctcttgctc aactggtact agcttgtagc
     2761 accatccaaa ggtcagtgga tatctgatcc ctggccctgg tgtgtagttc tgccaatcag
     2821 ggaagtagcc ttgtgtgtgg tagatccaca gatcaaggat atcttgtctt cgttgggagt
     2881 gaattagccc ttccagtccc cccttttctt ttaaaaagtg gctaagatct acagctgcct
     2941 tgtaagtcat tggtcttaaa ggtacctgag gtgtgactgg aaaacccacc tcctcctcct
     3001 cttgtgcttc tagccaggca caatcagcat tggtagctgc tgtattgcta cttgtgattg
     3061 ctccatgttt ttctaggtct cgaggtcgac ggtatcgatg cggggaggcg gcccaaaggg
     3121 agatccgact cgtctgaggg cgaaggcgaa gacgcggaag aggccgcaga gccggcagca
     3181 ggccgcggga aggaaggtcc gctggattga gggccgaagg gacgtagcag aaggacgtcc
     3241 cgcgcagaat ccaggtggca acacaggcga gcagccaagg aaaggacgat gatttccccg
     3301 acaacaccac ggaattgtca gtgcccaaca gccgagcccc tgtccagcag cgggcaaggc
     3361 aggcggcgat gagttccgcc gtggcaatag ggagggggaa agcgaaagtc ccggaaagga
     3421 gctgacaggt ggtggcaatg ccccaaccag tgggggttgc gtcagcaaac acagtgcaca
     3481 ccacgccacg ttgcctgaca acgggccaca actcctcata aagagacagc aaccaggatt
     3541 tatacaagga ggagaaaatg aaagccatac gggaagcaat agcatgatac aaaggcatta
     3601 aagcagcgta tccacatagc gtaaaaggag caacatagtt aagaatacca gtcaatcttt
     3661 cacaaatttt gtaatccaga ggttgattac cgataagctt gatatcgaat tcttagccct
     3721 cccacacata accagagggc agcaattcac gaatcccaac tgccgtcggc tgtccatcac
     3781 tgtccttcac tatggctttg atcccaggat gcagatcgag aagcacctgt cggcaccgtc
     3841 cgcaggggct caagatgccc ctgttctcat ttccgatcgc gacgatacaa gtcaggttgc
     3901 cagctgccgc agcagcagca gtgcccagca ccacgagttc tgcacaaggt cccccagtaa
     3961 aatgatatac attgacacca gtgaagatgc ggccgtcgct agagagagct gcgctggcga
     4021 cgctgtagtc ttcagagatg gggatgctgt tgattgtagc cgttgctctt tcaatgaggg
     4081 tggattcttc ttgagacaaa ggcttggcca ttgggccagg attctcctcg acgtcaccgc
     4141 atgttagcag acttcctctg ccctctccac tgcctgtaca gttaatcagc atgtccaggt
     4201 cgaaatcatc aagggcgtca ctgccgagca tgtcgaggtc aaagtcatca agggcatccg
     4261 aaccaagcat gtcaaggtca aaatcatcga gggcgtcact tcccagcata tccagatcaa
     4321 aatcgtccaa tgcgtcagcc cgtccggatc cagcggccgc caccttcctc tttttcttag
     4381 gtccgctacc tcctcctccg cttcctcctc ctccgcttcc acctcctcca gcgctgtcgc
     4441 ctcccagctg agacaggtcg atccgtgtct cgtacaggcc ggtgatgctc tggtggatca
     4501 gggtggcgtc cagcacctct ttggtgctgg tgtacctctt ccggtcgatg gtggtgtcaa
     4561 agtacttgaa ggcggcaggg gctcccagat tggtcagggt aaacaggtgg atgatattct
     4621 cggcctgctc tctgatgggc ttatcccggt gcttgttgta ggcggacagc actttgtcca
     4681 gattagcgtc ggccaggatc actctcttgg agaactcgct gatctgctcg atgatctcgt
     4741 ccaggtagtg cttgtgctgt tccacaaaca gctgtttctg ctcattatcc tcgggggagc
     4801 ccttcagctt ctcatagtgg ctggccaggt acaggaagtt cacatatttg gagggcaggg
     4861 ccagttcgtt tcccttctgc agttcgccgg cagaggccag cattctcttc cggccgtttt
     4921 ccagctcgaa cagggagtac ttaggcagct tgatgatcag gtcctttttc acttctttgt
     4981 agcccttggc ttccagaaag tcgatgggat tcttctcgaa gctgcttctt tccatgatgg
     5041 tgatccccag cagctctttc acactcttca gtttcttgga cttgcccttt tccactttgg
     5101 ccaccaccag cacagaatag gccacggtgg ggctgtcgaa gccgccgtac ttcttagggt
     5161 cccagtcctt ctttctggcg atcagcttat cgctgttcct cttgggcagg atagactctt
     5221 tgctgaagcc gcctgtctgc acctcggtct ttttcacgat attcacttgg ggcatgctca
     5281 gcactttccg cacggtggca aaatcccggc ccttatccca cacgatctcc ccggtttcgc
     5341 cgtttgtctc gatcagaggc cgcttccgga tctcgccgtt ggccagggta atctcggtct
     5401 tgaaaaagtt catgatgttg ctgtagaaga agtacttggc ggtagccttg ccgatttcct
     5461 gctcgctctt ggcgatcatc ttccgcacgt cgtacacctt gtagtcgccg tacacgaact
     5521 cgctttccag cttagggtac tttttgatca gggcggttcc cacgacggcg ttcaggtagg
     5581 cgtcgtgggc gtggtggtag ttgttgatct cgcgcacttt gtaaaactgg aaatccttcc
     5641 ggaaatcgga caccagcttg gacttcaggg tgatcacttt cacttcccgg atcagcttgt
     5701 cattctcgtc gtacttagtg ttcatccggg agtccaggat ctgtgccacg tgctttgtga
     5761 tctgccgggt ttccaccagc tgtctcttga tgaagccggc cttatccagt tcgctcaggc
     5821 cgcctctctc ggccttggtc agattgtcga actttctctg ggtaatcagc ttggcgttca
     5881 gcagctgccg ccagtagttc ttcatcttct tcacgacctc ttcggagggc acgttgtcgc
     5941 tcttgccccg ggccttgtcg cttctggtca gcaccttgtt gtcgatggag tcgtccttca
     6001 gaaagctctg aggcacgatg tggtccacat cgtagtcgga cagccggttg atgtccagtt
     6061 cctggtccac gtacatatcc cgcccattct gcaggtagta caggtacagc ttctcgttct
     6121 gcagctgggt gttttccacg gggtgttctt tcaggatctg gctgcccagc tctttgatgc
     6181 cctcttcgat ccgcttcatt ctctcgcggc tgttcttctg tcccttctgg gtggtctggt
     6241 tctctctggc catttcgatc acgatgttct cgggcttgtg ccggcccatc actttcacga
     6301 gctcgtccac caccttcact gtctgcagga tgcccttctt aatggcgggg ctgccggcca
     6361 gattggcaat gtgctcgtgc aggctatcgc cctggccgga cacctgggct ttctggatgt
     6421 cctctttaaa ggtcaggctg tcgtcgtgga tcagctgcat gaagtttctg ttggcgaagc
     6481 cgtcggactt caggaaatcc aggattgtct tgccggactg cttgtcccgg atgccgttga
     6541 tcagcttccg gctcagcctg ccccagccgg tgtatctccg ccgcttcagc tgcttcatca
     6601 ctttgtcgtc gaacaggtgg gcataggttt tcagccgttc ctcgatcatc tctctgtcct
     6661 caaacagtgt cagggtcagc acgatatctt ccagaatgtc ctcgttttcc tcattgtcca
     6721 ggaagtcctt gtccttgata attttcagca gatcgtggta tgtgcccagg gaggcgttga
     6781 accgatcttc cacgccggag atttccacgg agtcgaagca ctcgattttc ttgaagtagt
     6841 cctctttcag ctgcttcacg gtcactttcc ggttggtctt gaacagcagg tccacgatgg
     6901 cctttttctg ctcgccgctc aggaaggcgg gctttctcat tccctcggtc acgtatttca
     6961 ctttggtcag ctcgttatac acggtgaagt actcgtacag caggctgtgc ttgggcagca
     7021 ccttctcgtt gggcaggttc ttatcgaagt tggtcatccg ctcgatgaag ctctgggcgg
     7081 aagcgccctt gtccaccact tcctcgaagt tccagggggt gatggtttcc tcgctctttc
     7141 tggtcatcca ggcgaatctg ctgtttcccc tggccagagg gcccacgtag taggggatgc
     7201 ggaaggtcag gatcttctcg atcttttccc ggttgtcctt caggaatggg taaaaatctt
     7261 cctgccgccg cagaatggcg tgcagctctc ccaggtggat ctggtggggg atgctgccgt
     7321 tgtcgaaggt ccgctgcttc cgcagcaggt cctctctgtt cagcttcacg agcagttcct
     7381 cggtgccgtc catcttttcc aggatgggct tgatgaactt gtagaactct tcctggctgg
     7441 ctccgccgtc aatgtagccg gcgtagccgt tcttgctctg gtcgaagaaa atctctttgt
     7501 acttctcagg cagctgctgc cgcacgagag ctttcagcag ggtcaggtcc tggtggtgct
     7561 cgtcgtatct cttgatcata gaggcgctca ggggggcctt ggtgatctcg gtgttcactc
     7621 tcaggatgtc gctcagcagg atggcgtcgg acaggttctt ggcggccaga aacaggtcgg
     7681 cgtactggtc gccgatctgg gccagcaggt tgtccaggtc gtcgtcgtag gtgtccttgc
     7741 tcagctgcag tttggcatcc tcggccaggt cgaagttgct cttgaagttg ggggtcaggc
     7801 ccaggctcag ggcaatcagg ttgccgaaca ggccattctt cttctcgccg ggcagctggg
     7861 cgatcagatt ttccagccgt ctgctcttgc tcagtctggc agacaggatg gccttggcgt
     7921 ccacgccgct ggcgttgatg gggttttcct cgaacagctg gttgtaggtc tgcaccagct
     7981 ggatgaacag cttgtccacg tcgctgttgt cggggttcag gtcgccctcg atcaggaagt
     8041 ggccccggaa cttgatcatg tgggccaggg ccagatagat cagccgcagg tcggccttgt
     8101 cggtgctgtc caccagtttc tttctcaggt ggtagatggt ggggtacttc tcgtggtagg
     8161 ccacctcgtc cacgatgttg ccgaagatgg ggtgccgctc gtgcttctta tcctcttcca
     8221 ccaggaagga ctcttccagt ctgtggaaga agctgtcgtc caccttggcc atctcgttgc
     8281 tgaagatctc ttgcagatag cagatccggt tcttccgtct ggtgtatctt cttctggcgg
     8341 ttctcttcag ccgggtggcc tcggctgttt cgccgctgtc gaacagcagg gctccgatca
     8401 ggttcttctt gatgctgtgc cggtcggtgt tgcccagcac cttgaatttc ttgctgggca
     8461 ccttgtactc gtcggtgatc acggcccagc ccacagagtt ggtgccgatg gccaggccga
     8521 tgctgtactt cttgtccttt ttcttttttg cctggccggc ctttttcgtg gccgccggcc
     8581 ttttcatggt ggccgtacgc ctgtgttctg gcggcaaacc cgttgcgaaa aagaacgttc
     8641 acggcgacta ctgcacttat atacggttct cccccaccct cgggaaaaag gcggagccag
     8701 tacacgacat cactttccca gtttaccccg cgccaccttc tctaggcacc ggatcaattg
     8761 ccgacccctc cccccaactt ctcggggact gtgggcgatg tgcgctctgc ccactgacgg
     8821 gcaccggagc caattcccac tcctttcaag acctaggatc caaaaaaagc accgactcgg
     8881 tgccacttgg ccctgcagac atgggtgatc ctcatgttgg ccaagttgat aacggactag
     8941 ccttatttta acttgctagg ccctgcagac atgggtgatc ctcatgttgg cctagctcta
     9001 aaacagagac ggtatcccgt ctccggtgtt tcgtcctttc cacaagatat ataaagccaa
     9061 gaaatcgaaa tactttcaag ttacggtaag catatgatag tccattttaa aacataattt
     9121 taaaactgca aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat
     9181 ctttgtgttt acagtcaaat taattccaat tatctctcta acagccttgt atcgtatatg
     9241 caaatatgaa ggaatcatgg gaaataggcc ctcgctagct aattaaccaa actggatctc
     9301 tgctgtccct gtaataaacc cgaaaatttt gaatttttgt aatttgtttt tgtaattctt
     9361 tagtttgtat gtctgttgct attatgtcta ctattctttc ccctgcactg taccccccaa
     9421 tccccccttt tcttttaaaa ttgtggatga atactgccat ttgtctgcag aattggcgca
     9481 cgcagtgccg atccgttcac taatcgaatg gatctgtctc tgtctctctc tccaccttct
     9541 tcttctattc cttcgggcct gtcgggtccc ctcggggttg ggaggtgggt ctgaaacgat
     9601 aatggtgaat atccctgcct aactctattc actatagaaa gtacagcaaa aactattctt
     9661 aaacctacca agcctcctac tatcattatg aataatttta tataccacag ccaatttgtt
     9721 atgttaaacc aattccacaa acttgcccat ttatctaatt ccaataattc ttgttcattc
     9781 ttttcttgct ggttttgcga ttcttcaatt aaggagtgta ttaagcttgt gtaattgtta
     9841 atttctctgt cccactccat ccaggtcgtg tgattccaaa tctgttccag agatttatta
     9901 ctccaactag cattccaagg cacagcagtg gtgcaaatga gttttccaga gcaaccccaa
     9961 atccccagga gctgttgatc ctttaggtat ctttccacag ccaggattct tgcctggagc
    10021 tgcttgatgc cccagactgt gagttgcaac agatgctgtt gcgcctcaat agccctcagc
    10081 aaattgttct gctgctgcac tataccagac aataattgtc tggcctgtac cgtcagcgtc
    10141 attgacgctg cgcccatagt gcttcctgct gctcccaaga acccaaggaa caaagctcct
    10201 attcccactg ctcttttttc tctctgcacc actcttctct ttgccttggt gggtgctact
    10261 cctaatggtt caatttttac tactttatat ttatataatt cacttctcca attgtccctc
    10321 atatctcctc ctccaggtct gaagatcagc ggccgcttgc tgtgcggtgg tcttactttt
    10381 gttttgctct tcctctatct tgtctaaagc ttccttggtg tcttttatct ctatcctttg
    10441 atgcacacaa tagagggttg ctactgtatt atataatgat ctaagttctt ctgatcctgt
    10501 ctgaagggat ggttgtagct gtcccagtat ttgtctacag ccttctgatg tttctaacag
    10561 gccaggatta actgcgaatc gttctagctc cctgcttgcc catactatat gttttaattt
    10621 atattttttc tttccccctg gccttaaccg aattttttcc catcgcgatc taattctccc
    10681 ccgcttaata ctgacgctct cgcacccatc tctctccttc tagcctccgc tagtcaaaat
    10741 ttttggcgta ctcaccagtc gccgcccctc gcctcttgcc gtgcgcgctt cagcaagccg
    10801 agtcctgcgt cgagagagct cctctggttt ccctttcgct ttcaagtccc tgttcgggcg
    10861 ccactgctag agattttcca cactgactaa aagggtctga gggatctcta gttaccagag
    10921 tcacacaaca gacgggcaca cactacttga agcactcaag gcaagcttta ttgaggctta
    10981 agcagtgggt tccctagtta gccagagagc tcccaggctc agatctggtc taaccagaga
    11041 gacccagtac aggcaaaacg cgctgcttat atagacctcc caccgtacac gcctaccgcc
    11101 catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg
    11161 ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa
    11221 ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac
    11281 taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg
    11341 ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac
    11401 ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa
    11461 agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc
    11521 gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata
    11581 tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg
    11641 tcaacgcgta tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag
    11701 attcttcatg caattgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca
    11761 ctactcagcg acctccaaca cacaagcagg gagcagatac tggcttaact atgcggcatc
    11821 agagcagatt gtactgagag tgcaccatag gggatcggga gatctcccga tccgtcgacg
    11881 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
    11941 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
    12001 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
    12061 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
    12121 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
    12181 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
    12241 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
    12301 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
    12361 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
    12421 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
    12481 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
    12541 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
    12601 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
    12661 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
    12721 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
    12781 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
    12841 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
    12901 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
    12961 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
    13021 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
    13081 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
    13141 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
    13201 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
    13261 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
    13321 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
    13381 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
    13441 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
//
