LOCUS       sequence-267569-       11785 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Lentiviral expression of AsCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11785)
  AUTHORS   DeWeirdt PC, Sanson KR, Sangree AK, Hegde M, Hanna RE, Feeley MN, 
            Griffith AL, Teng T, Borys SM, Strand C, Joung JK, Kleinstiver BP, 
            Pan X, Huang A, Doench JG
  TITLE     Optimization of AsCas12a for combinatorial genetic screens in human 
            cells.
  JOURNAL   Nat Biotechnol. 2020 Jul 13. pii: 10.1038/s41587-020-0600-6. doi: 
            10.1038/s41587-020-0600-6.
  PUBMED    32661438
REFERENCE   2  (bases 1 to 11785)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2020 Jul 13. pii: 10.1038/s41587-020-0600-6. doi: 
            10.1038/s41587-020-0600-6."
COMMENT     Sequence Label: pRDA_112
FEATURES             Location/Qualifiers
     source          1..11785
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        32..258
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             259..439
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    486..611
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1104..1337
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1522..1566
                     /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    1844..1959
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     promoter        2111..3288
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2342..3279
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3236..3256
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3297..3306
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3309..3329
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3354..7271
                     /codon_start=1
                     /product="CRISPR-associated protein from Acidaminococcus 
                     sp. BV3L6 (Zetsche et al., 2015)"
                     /label=AsCpf1
                     /translation="TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH
                     YKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNA
                     IHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDK
                     FTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE
                     NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLN
                     LAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLR
                     NENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGK
                     ITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLK
                     KQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKAR
                     NYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKA
                     LSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPL
                     EITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDL
                     SSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGH
                     HGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKD
                     QKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFH
                     VPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRS
                     LNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVV
                     VLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQF
                     TSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDV
                     KTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENH
                     RFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMR
                     NSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESK
                     DLKLQNGISNQDWLAYIQELRN"
     regulatory      6991..7000
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7272..7319
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7326..7406
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             7422..7475
                     /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             7482..7880
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    7890..8478
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7943..7963)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8361..8372)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8552..8785
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    8857..8978
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8894..8913)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8948..8967
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      9018..9153
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(9186..9202)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9186..9202)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9199..9221)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9210..9226
                     /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(9234..9240,9241..9258,9259..9264))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9279..9300
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9417..9434)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9588..10176)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(10347..11138,11139..11207))
                     /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     10970..10989
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11208..11312)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     11380..11398
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(11436..11458)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     11558..11577
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     join(11771..11785,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(11785,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgcc aagctgacgc gtgtagtctt atgcaatact cttgtagtct
       61 tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca
      121 tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt
      181 ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc
      241 ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
      301 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
      361 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
      421 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
      481 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
      541 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
      601 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
      661 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
      721 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
      781 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
      841 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
      901 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccactgatc
      961 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa
     1021 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag
     1081 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga
     1141 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt
     1201 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa
     1261 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta
     1321 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct
     1381 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc
     1441 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa
     1501 gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca
     1561 agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg
     1621 atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga
     1681 gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc
     1741 gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag atccattcga
     1801 ttagtgaacg gatctcgacg gtatcgttgc ttggatcgag caatttaaaa gaaaaggggg
     1861 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     1921 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttc tgcgttgttg tcggtgctcg
     1981 ttctctgctc ttcacgctac tgaattcatc accggttctt cgaatctagt tgcaaagatg
     2041 gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg tcttgaaagg
     2101 agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
     2161 gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa
     2221 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
     2281 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
     2341 ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt
     2401 gccttgaatt acttccactg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     2461 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     2521 gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     2581 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     2641 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
     2701 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
     2761 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     2821 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     2881 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     2941 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     3001 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     3061 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt
     3121 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     3181 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     3241 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgatt ctagccgcca
     3301 ccatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccacacagt
     3361 tcgagggctt taccaacctg tatcaggtga gcaagacact gcggtttgag ctgatcccac
     3421 agggcaagac cctgaagcac atccaggagc agggcttcat cgaggaggac aaggcccgca
     3481 atgatcacta caaggagctg aagcccatca tcgatcggat ctacaagacc tatgccgacc
     3541 agtgcctgca gctggtgcag ctggattggg agaacctgag cgccgccatc gactcctata
     3601 gaaaggagaa aaccgaggag acaaggaacg ccctgatcga ggagcaggcc acatatcgca
     3661 atgccatcca cgactacttc atcggccgga cagacaacct gaccgatgcc atcaataaga
     3721 gacacgccga gatctacaag ggcctgttca aggccgagct gtttaatggc aaggtgctga
     3781 agcagctggg caccgtgacc acaaccgagc acgagaacgc cctgctgcgg agcttcgaca
     3841 agtttacaac ctacttctcc ggcttttatg agaacaggaa gaacgtgttc agcgccgagg
     3901 atatcagcac agccatccca caccgcatcg tgcaggacaa cttccccaag tttaaggaga
     3961 attgtcacat cttcacacgc ctgatcaccg ccgtgcccag cctgcgggag cactttgaga
     4021 acgtgaagaa ggccatcggc atcttcgtga gcacctccat cgaggaggtg ttttccttcc
     4081 ctttttataa ccagctgctg acacagaccc agatcgacct gtataaccag ctgctgggag
     4141 gaatctctcg ggaggcaggc accgagaaga tcaagggcct gaacgaggtg ctgaatctgg
     4201 ccatccagaa gaatgatgag acagcccaca tcatcgcctc cctgccacac agattcatcc
     4261 ccctgtttaa gcagatcctg tccgatagga acaccctgtc tttcatcctg gaggagttta
     4321 agagcgacga ggaagtgatc cagtccttct gcaagtacaa gacactgctg agaaacgaga
     4381 acgtgctgga gacagccgag gccctgttta acgagctgaa cagcatcgac ctgacacaca
     4441 tcttcatcag ccacaagaag ctggagacaa tcagcagcgc cctgtgcgac cactgggata
     4501 cactgaggaa tgccctgtat gagcggagaa tctccgagct gacaggcaag atcaccaagt
     4561 ctgccaagga gaaggtgcag cgcagcctga agcacgagga tatcaacctg caggagatca
     4621 tctctgccgc aggcaaggag ctgagcgagg ccttcaagca gaaaaccagc gagatcctgt
     4681 cccacgcaca cgccgccctg gatcagccac tgcctacaac cctgaagaag caggaggaga
     4741 aggagatcct gaagtctcag ctggacagcc tgctgggcct gtaccacctg ctggactggt
     4801 ttgccgtgga tgagtccaac gaggtggacc ccgagttctc tgcccggctg accggcatca
     4861 agctggagat ggagccttct ctgagcttct acaacaaggc cagaaattat gccaccaaga
     4921 agccctactc cgtggagaag ttcaagctga actttcagat gcctacactg gcctctggct
     4981 gggacgtgaa taaggagaag aacaatggcg ccatcctgtt tgtgaagaac ggcctgtact
     5041 atctgggcat catgccaaag cagaagggca ggtataaggc cctgagcttc gagcccacag
     5101 agaaaaccag cgagggcttt gataagatgt actatgacta cttccctgat gccgccaaga
     5161 tgatcccaaa gtgcagcacc cagctgaagg ccgtgacagc ccactttcag acccacacaa
     5221 cccccatcct gctgtccaac aatttcatcg agcctctgga gatcacaaag gagatctacg
     5281 acctgaacaa tcctgagaag gagccaaaga agtttcagac agcctacgcc aagaaaaccg
     5341 gcgaccagaa gggctacaga gaggccctgt gcaagtggat cgacttcaca agggattttc
     5401 tgtccaagta taccaagaca acctctatcg atctgtctag cctgcggcca tcctctcagt
     5461 ataaggacct gggcgagtac tatgccgagc tgaatcccct gctgtaccac atcagcttcc
     5521 agagaatcgc cgagaaggag atcatggatg ccgtggagac aggcaagctg tacctgttcc
     5581 agatctataa caaggacttt gccaagggcc accacggcaa gcctaatctg cacacactgt
     5641 attggaccgg cctgttttct ccagagaacc tggccaagac aagcatcaag ctgaatggcc
     5701 aggccgagct gttctaccgc cctaagtcca ggatgaagag gatggcacac cggctgggag
     5761 agaagatgct gaacaagaag ctgaaggatc agaaaacccc aatccccgac accctgtacc
     5821 aggagctgta cgactatgtg aatcacagac tgtcccacga cctgtctgat gaggccaggg
     5881 ccctgctgcc caacgtgatc accaaggagg tgtctcacga gatcatcaag gataggcgct
     5941 ttaccagcga caagttcttt ttccacgtgc ctatcacact gaactatcag gccgccaatt
     6001 ccccatctaa gttcaaccag agggtgaatg cctacctgaa ggagcacccc gagacaccta
     6061 tcatcggcat cgatcggggc gagagaaacc tgatctatat cacagtgatc gactccaccg
     6121 gcaagatcct ggagcagcgg agcctgaaca ccatccagca gtttgattac cagaagaagc
     6181 tggacaacag ggagaaggag agggtggcag caaggcaggc ctggtctgtg gtgggcacaa
     6241 tcaaggatct gaagcagggc tatctgagcc aggtcatcca cgagatcgtg gacctgatga
     6301 tccactacca ggccgtggtg gtgctggaga acctgaattt cggctttaag agcaagagga
     6361 ccggcatcgc cgagaaggcc gtgtaccagc agttcgagaa gatgctgatc gataagctga
     6421 attgcctggt gctgaaggac tatccagcag agaaagtggg aggcgtgctg aacccatacc
     6481 agctgacaga ccagttcacc tcctttgcca agatgggcac ccagtctggc ttcctgtttt
     6541 acgtgcctgc cccatataca tctaagatcg atcccctgac cggcttcgtg gaccccttcg
     6601 tgtggaaaac catcaagaat cacgagagcc gcaagcactt cctggagggc ttcgactttc
     6661 tgcactacga cgtgaaaacc ggcgacttca tcctgcactt taagatgaac agaaatctgt
     6721 ccttccagag gggcctgccc ggctttatgc ctgcatggga tatcgtgttc gagaagaacg
     6781 agacacagtt tgacgccaag ggcacccctt tcatcgccgg caagagaatc gtgccagtga
     6841 tcgagaatca cagattcacc ggcagatacc gggacctgta tcctgccaac gagctgatcg
     6901 ccctgctgga ggagaagggc atcgtgttca gggatggctc caacatcctg ccaaagctgc
     6961 tggagaatga cgattctcac gccatcgaca ccatggtggc cctgatccgc agcgtgctgc
     7021 agatgcggaa ctccaatgcc gccacaggcg aggactatat caacagcccc gtgcgcgatc
     7081 tgaatggcgt gtgcttcgac tcccggtttc agaacccaga gtggcccatg gacgccgatg
     7141 ccaatggcgc ctaccacatc gccctgaagg gccagctgct gctgaatcac ctgaaggaga
     7201 gcaaggatct gaagctgcag aacggcatct ccaatcagga ctggctggcc tacatccagg
     7261 agctgcgcaa caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg
     7321 gatcctaccc atacgatgtt ccagattacg cttatcccta cgacgtgcct gattatgcat
     7381 acccatatga tgtccccgac tatgccgcgg ccgcaggatg cgagggcaga ggaagccttc
     7441 taacatgcgg tgacgtggag gagaatcccg gcccttccgg gatggccaag cctttgtctc
     7501 aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg
     7561 aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca
     7621 atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg
     7681 ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct
     7741 tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca
     7801 tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg
     7861 gttatgtgtg ggagggctaa taagtcgaca atcaacctct ggattacaaa atttgtgaaa
     7921 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     7981 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     8041 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     8101 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     8161 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     8221 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     8281 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     8341 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     8401 tgccggctct gcggcctctt ccgcgtctcc gccttcgccc tcagacgagt cggatctctc
     8461 tttgggccgc ctccccgcct ggtaccttta agaccaatga cttacaaggc agctgtagat
     8521 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     8581 taagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     8641 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     8701 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     8761 tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat tcagtattta
     8821 taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat
     8881 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     8941 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc
     9001 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     9061 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     9121 tccagaagta gtgaggaggc ttttttggag gcctagactt ttgcagagac caaattcgta
     9181 atcatgtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     9241 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     9301 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     9361 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     9421 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     9481 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     9541 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     9601 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     9661 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     9721 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     9781 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     9841 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     9901 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     9961 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
    10021 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
    10081 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
    10141 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
    10201 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
    10261 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
    10321 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
    10381 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
    10441 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
    10501 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
    10561 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
    10621 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
    10681 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
    10741 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
    10801 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
    10861 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
    10921 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
    10981 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
    11041 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
    11101 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
    11161 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
    11221 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
    11281 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
    11341 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
    11401 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
    11461 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
    11521 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
    11581 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
    11641 aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
    11701 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
    11761 ccagggtttt cccagtcacg acgtt
//
