CROPseq-Guide-Puro

LOCUS       CROPseq-Guide-Puro               10214 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Makes CRISPR gRNAs detectable by single-cell RNA-seq. The gRNA is 
            expressed as part of the puromycin resistance mRNA transcribed by 
            RNA Pol II and in its functional form from the hU6 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    CROPseq-Guide-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10214)
  AUTHORS   Datlinger P, Rendeiro AF, Schmidl C, Krausgruber T, Traxler P, 
            Klughammer J, Schuster LC, Kuchler A, Alpar D, Bock C
  TITLE     Pooled CRISPR screening with single-cell transcriptome readout.
  JOURNAL   Nat Methods. 2017 Jan 18. doi: 10.1038/nmeth.4177.
  PUBMED    28099430
REFERENCE   2  (bases 1 to 10214)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..10214
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(168..756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(248..267)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(927..1787)
                     /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     1550..1569
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1788..1892)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1918..2373)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2055..2076)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2267..2286
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2500..2522
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2514..2531
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2515..2531
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2541..2560
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2541..2559
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      2580..2715
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2634..2653)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2755..2876
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2766..2785)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2820..2839
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             2954..3134
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_RNA        complement(3187..3262)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(5152..5392)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     complement(5202..5221)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5372..5392)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    5548..6136
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             5654..5665
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     6063..6083
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6152..6751)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6235..6255)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     6732..6751
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(6764..7942)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          6773..7711
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(6796..6816)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     misc_feature    8081..8198
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(8379..8423)
                     /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    8608..8841
                     /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    9334..9459
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             9506..9686
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(9687..9913)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(9941..9961)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(9941..9959)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9980..9996)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9980..9996)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9993..10015)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10004..10020
                     /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(10028..10058)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10073..10094
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10211..14)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
       61 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
      121 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
      181 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      241 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      301 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      361 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      421 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
      481 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
      541 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
      601 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
      661 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
      721 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
      781 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
      841 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
      901 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
      961 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     1021 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     1081 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     1141 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     1201 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     1261 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     1321 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     1381 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     1441 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     1501 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     1561 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     1621 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     1681 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     1741 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     1801 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     1861 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     1921 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     1981 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     2041 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     2101 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     2161 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     2221 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2281 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2341 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
     2401 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     2461 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     2521 cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggt acgtccctag
     2581 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg cggcctcggc
     2641 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     2701 agttaggggc gggatgggcg gagttagggg cgggatagct agagccagac atgataagat
     2761 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     2821 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttcctc
     2881 tcactctctg atattcattt ctttgcaagt tataaatact gaataataag atgacatgaa
     2941 ctactatacg tactgctaga gattttccac actgactaaa agggtctgag ggatctctag
     3001 ttaccagagt cacacaacag acgggcacac actacttgaa gcactcaagg caagctttat
     3061 tgaggcttaa gcagtgggtt ccctagttag ccagagagct cccaggctca gatctggtct
     3121 aaccagagag acccagtaca agcaaaaagc agtgtctcaa gatctagtta cgccaagctt
     3181 aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac
     3241 ttgctatttc tagctctaaa acagagacgt acaaaaaaga gcaagaagct aaaaaagatt
     3301 taaaaattat ttttagcgca gttaatggaa caggaactaa atttacccca aaaatattac
     3361 gtgaatcagg atataacgtt attgaggttg aagagcatgc atttgaagat gaaacattta
     3421 aaaatgttgt aaatccaaat ccagaatttg atcctgcatg aaaaataccg cttgaatatg
     3481 gtattaaaca tgatgcagat attattatta tgaatgaccc agatgctgac agatttggaa
     3541 tggcaataaa acatgatggt cattttgtaa gattagatgg aaatcaaaca ggaccaattt
     3601 taattgattg aaaattatca aatctaaaac gcttaaatag cattccaaaa aatccggctc
     3661 tatattcaag ttttgtaaca agtgatttgg gtgatagaat cgctcatgaa aaatatggag
     3721 ttaatattgt aaaaacttta actggattta aatgaatggg tagagaaatt gctaaagaag
     3781 aagataacgg attaaatttt gtttttgctt atgaagaaag ttatggatat gtaattgatg
     3841 actcagctag agataaagat ggaatacaag cttctatatt aatagcagag gctgcttgat
     3901 tttataaaaa acaaaataaa acattagtag actatttaga agatttattt aaagaaatgg
     3961 gtgcatatta cactttcact ttaaacttga attttaaacc agaagaaaag aaattaaaaa
     4021 ttgaaccatt aatgaaatca ttgagagcaa cacccttaac tcaaattgct ggacttaaag
     4081 ttgttaatgt tgaagactac atcgatggaa tgtataatat gccaggacaa gacttactaa
     4141 aattttattt agaagataag tcatgatttg ctgttcgccc aagtggaact gaacctaaac
     4201 taaaaattta ttttataggt gttggtgaat ctgttcaaaa cgctaaagtt aaagtagacg
     4261 aaattattaa agaattaaaa ttaaaaatga atatatagga gaaaaaatga aactaaacaa
     4321 atatatagat cacacattat taaaacaaga tgctacgaaa gctgaaatta aacaattatg
     4381 tgatgaagca attgaatttg attttgcaac agtttgtgtt aattcatatt gaacaagcta
     4441 ttgtaaagaa ttattaaaag gcacaaatgt aggaataaca aatgttgtag gttttcctct
     4501 aggtgcatgc acaacagcta caaaagcatt cgaagtttct gaagcaatta aagatggtgc
     4561 aacagaaatt gatatggtat taaatattgg tgcattaaaa gacaaaaatt atgaattagt
     4621 tttagaagac atgaaagctg taaaaaaagc agctggatca catgttgtta aatgtattat
     4681 ggaaaattgt ttattaacaa aagaagaaat catgaaagct tgtgaaatag ctgttgaagc
     4741 tggattagaa tttgttaaaa catcaacagg attttcaaaa tcaggtgcaa catttgaaga
     4801 tgttaaacta atgaagtcag ttgttaaaga caatgcttta gttaaagcag ctggtggagt
     4861 tagaacattt gaagatgctc aaaaaatgat tgaagcagga gctgaccgct taggaacaag
     4921 tggtggagta gctattatta aaggtgaaga aaacaacgcg agttactaaa actagcgttt
     4981 ttttattttg ctcattttta ttaaaagttt gcaaaaagga acataaaaat tctaattatt
     5041 gatactaaag ttattaaaaa gaagattttg gttgatttta taaaggtcat agaatataat
     5101 attttagcat gtgtattttg tgtgctcatt tacaaccgtc tccggtgttt cgtcctttcc
     5161 acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc atatgatagt
     5221 ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac tttctacgtc
     5281 acgtattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt atctctctaa
     5341 cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc tctctgggtc
     5401 ccctcggggt tgggaggtgg gtctgaaacg ataatggtga aatcttgtct tcgttgggag
     5461 tgaattagcc cttccagtcc ccccttttct tttaaaaagt ggctaagatc tacagctgcc
     5521 ttgtaagtca ttggtcttaa agtcgacgcg gggaggcggc ccaaagggag atccgactcg
     5581 tctgagggcg aaggcgaaga cgcggaagag gccgcagagc cggcagcagg ccgcgggaag
     5641 gaaggtccgc tggattgagg gccgaaggga cgtagcagaa ggacgtcccg cgcagaatcc
     5701 aggtggcaac acaggcgagc agccaaggaa aggacgatga tttccccgac aacaccacgg
     5761 aattgtcagt gcccaacagc cgagcccctg tccagcagcg ggcaaggcag gcggcgatga
     5821 gttccgccgt ggcaataggg agggggaaag cgaaagtccc ggaaaggagc tgacaggtgg
     5881 tggcaatgcc ccaaccagtg ggggttgcgt cagcaaacac agtgcacacc acgccacgtt
     5941 gcctgacaac gggccacaac tcctcataaa gagacagcaa ccaggattta tacaaggagg
     6001 agaaaatgaa agccatacgg gaagcaatag catgatacaa aggcattaaa gcagcgtatc
     6061 cacatagcgt aaaaggagca acatagttaa gaataccagt caatctttca caaattttgt
     6121 aatccagagg ttgattgtcg acttaacgcg ttcaggcacc gggcttgcgg gtcatgcacc
     6181 aggtgcgcgg tccttcgggc acctcgacgt cggcggtgac ggtgaagccg agccgctcgt
     6241 agaaggggag gttgcggggc gcggaggtct ccaggaaggc gggcaccccg gcgcgctcgg
     6301 ccgcctccac tccggggagc acgacggcgc tgcccagacc cttgccctgg tggtcgggcg
     6361 agactccgac ggtggccagg aaccacgcgg gctccttggg ccggtgcggc gccaggaggc
     6421 cttccatctg ttgctgcgcg gccagccggg aaccgctcaa ctcggccatg cgcgggccga
     6481 tctcggcgaa caccgccccc gcttcgacgc tctccggcgt ggtccagacc gccacggcgg
     6541 cgccgtcgtc cgcgacccac accttgccga tgtcgagccc gacgcgcgtg aggaagagtt
     6601 cttgcagctc ggtgacccgc tcgatgtggc ggtccggatc gacggtgtgg cgcgtggcgg
     6661 ggtagtcggc gaacgcggcg gcgagggtgc gtacggccct ggggacgtcg tcgcgggtgg
     6721 cgaggcgcac cgtgggcttg tactcggtca tggtggccgt acgtcacgac acctgaaatg
     6781 gaagaaaaaa actttgaacc actgtctgag gcttgagaat gaaccaagat ccaaactcaa
     6841 aaagggcaaa ttccaaggag aattacatca agtgccaagc tggcctaact tcagtctcca
     6901 cccactcagt gtggggaaac tccatcgcat aaaacccctc cccccaacct aaagacgacg
     6961 tactccaaaa gctcgagaac taatcgaggt gcctggacgg cgcccggtac tccgtggagt
     7021 cacatgaagc gacggctgag gacggaaagg cccttttcct ttgtgtgggt gactcacccg
     7081 cccgctctcc cgagcgccgc gtcctccatt ttgagctccc tgcagcaggg ccgggaagcg
     7141 gccatctttc cgctcacgca actggtgccg accgggccag ccttgccgcc cagggcgggg
     7201 cgatacacgg cggcgcgagg ccaggcacca gagcaggccg gccagcttga gactaccccc
     7261 gtccgattct cggtggccgc gctcgcaggc cccgcctcgc cgaacatgtg cgctgggacg
     7321 cacgggcccc gtcgccgccc gcggccccaa aaaccgaaat accagtgtgc agatcttggc
     7381 ccgcatttac aagactatct tgccagaaaa aaagcgtcgc agcaggtcat caaaaatttt
     7441 aaatggctag agacttatcg aaagcagcga gacaggcgcg aaggtgccac cagattcgca
     7501 cgcggcggcc ccagcgccca ggccaggcct caactcaagc acgaggcgaa ggggctcctt
     7561 aagcgcaagg cctcgaactc tcccacccac ttccaacccg aagctcggga tcaagaatca
     7621 cgtactgcag ccaggtggaa gtaattcaag gcacgcaagg gccataaccc gtaaagaggc
     7681 caggcccgcg ggaaccacac acggcactta cctgtgttct ggcggcaaac ccgttgcgaa
     7741 aaagaacgtt cacggcgact actgcactta tatacggttc tcccccaccc tcgggaaaaa
     7801 ggcggagcca gtacacgaca tcactttccc agtttacccc gcgccacctt ctctaggcac
     7861 cggatcaatt gccgacccct ccccccaact tctcggggac tgtgggcgat gtgcgctctg
     7921 cccactgacg ggcaccggag ccaattccca ctcctttcaa gacctagaag gtccattagc
     7981 tgcaaagatt cctctctgtt taaaacttta tccatctttg cacccgggcc ccctcgagcc
     8041 ggcgccaaag tggatctctg ctgtccctgt aataaacccg aaaattttga atttttgtaa
     8101 tttgtttttg taattcttta gtttgtatgt ctgttgctat tatgtctact attctttccc
     8161 ctgcactgta ccccccaatc cccccttttc ttttaaaatt gtggatgaat actgccattt
     8221 gtctcaagat tctgaaacga taatggtgaa tatccctgcc taactctatt cactatagaa
     8281 agtacagcaa aaactattct taaacctacc aagcctccta ctatcattat gaataatttt
     8341 atataccaca gccaatttgt tatgttaaac caattccaca aacttgccca tttatctaat
     8401 tccaataatt cttgttcatt cttttcttgc tggttttgcg attcttcaat taaggagtgt
     8461 attaagcttg tgtaattgtt aatttctctg tcccactcca tccaggtcgt gtgattccaa
     8521 atctgttcca gagatttatt actccaacta gcattccaag gcacagcagt ggtgcaaatg
     8581 agttttccag agcaacccca aatccccagg agctgttgat cctttaggta tctttccaca
     8641 gccaggattc ttgcctggag ctgcttgatg ccccagactg tgagttgcaa cagatgctgt
     8701 tgcgcctcaa tagccctcag caaattgttc tgctgctgca ctataccaga caataattgt
     8761 ctggcctgta ccgtcagcgt cattgacgct gcgcccatag tgcttcctgc tgctcccaag
     8821 aacccaagga acaaagctcc tattcccact gctctttttt ctctctgcac cactcttctc
     8881 tttgccttgg tgggtgctac tcctaatggt tcaattttta ctactttata tttatataat
     8941 tcacttctcc aattgtccct catatctcct cctccaggtc tgaagatcag cggccgcttg
     9001 ctgtgcggtg gtcttacttt tgttttgctc ttcctctatc ttgtctaaag cttccttggt
     9061 gtcttttatc tctatccttt gatgcacaca atagagggtt gctactgtat tatataatga
     9121 tctaagttct tctgatcctg tctgaaggga tggttgtagc tgtcccagta tttgtctaca
     9181 gccttctgat gtttctaaca ggccaggatt aactgcgaat cgttctagct ccctgcttgc
     9241 ccatactata tgttttaatt tatatttttt ctttccccct ggccttaacc gaattttttc
     9301 ccatcgcgat ctaattctcc cccgcttaat actgacgctc tcgcacccat ctctctcctt
     9361 ctagcctccg ctagtcaaaa tttttggcgt actcaccagt cgccgcccct cgcctcttgc
     9421 cgtgcgcgct tcagcaagcc gagtcctgcg tcgagagagc tcctctggtt tccctttcgc
     9481 tttcaagtcc ctgttcgggc gccactgcta gagattttcc acactgacta aaagggtctg
     9541 agggatctct agttaccaga gtcacacaac agacgggcac acactacttg aagcactcaa
     9601 ggcaagcttt attgaggctt aagcagtggg ttccctagtt agccagagag ctcccaggct
     9661 cagatctggt ctaaccagag agacccgttt atgtatcgag ctaggcactt aaatacaata
     9721 tctctgcaat gcggcaattc agtggttcgt ccaatccatg tcagacccgt ctgttgcctt
     9781 cctaataagg cacgatcgta ccaccttact tccaccaatc ggcatgcacg gtgctttttc
     9841 tctccttgta aggcatgttg ctaactcatc gttaccatgt tgcaagacta caagagtatt
     9901 gcataagact acattaagct tgcagctcca gcttttgttc cctttagtga gggttaattg
     9961 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
    10021 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
    10081 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
    10141 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
    10201 cttccgcttc ctcg
//