LOCUS       62056_11910        8419 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8419)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    34..167
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(204..220)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(228..244)
                     /label=lac operator
                     /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(252..282)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(297..318)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(606..1194)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1368..2225)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(2226..2330)
                     /label=AmpR promoter
     promoter        2964..3175
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             3188..3456
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human
                     T-cell leukemia virus (HTLV) type 1"
     CDS             3525..7628
                     /codon_start=1
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /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             7671..7724
                     /codon_start=1
                     /label=T2A
                     /note="2A peptide from Thosea asigna virus capsid protein"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             7731..8327
                     /codon_start=1
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
ORIGIN
        1 acgcgtctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
       61 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
      121 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
      181 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
      241 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
      301 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
      361 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
      421 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
      481 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
      541 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
      601 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      661 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      721 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      781 ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      841 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
      901 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
      961 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     1021 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     1081 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     1141 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     1201 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     1261 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     1321 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     1381 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     1441 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     1501 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     1561 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     1621 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     1681 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     1741 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     1801 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     1861 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     1921 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     1981 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     2041 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     2101 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     2161 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     2221 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     2281 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     2341 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtcgactc
     2401 tcagtacaat ctgctctgat gccgcatgct aatgccaact ttgtacaaga aagctgggtc
     2461 tagaaaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt
     2521 taacttaaga tatataaagc caagaaatcg aaatactttc aagttacggt aagcatatga
     2581 tagtccattt taaaacataa ttttaaaact gcaaactacc caagaaatta ttactttcta
     2641 cgtcacgtat tttgtactaa tatctttgtg tttacagtca aattaattct aattatctct
     2701 ctaacagcct tgtatcgtat atgcaaatat gaaggaatca tgggaaatag gccctcttcc
     2761 tgcccgacct tggtaccgga tccagtcgac tgaattggtt cctttaaagc ctgctttttt
     2821 gtacatgcat ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     2881 actaaagaat tacaaaaaca aattacaaaa ttcaaaattt tatcgatact agtggatctg
     2941 cgatcgctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt
     3001 ggggggaggg gtcggcaatt gaacgggtgc ctagagaagg tggcgcgggg taaactggga
     3061 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
     3121 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacagctgaa
     3181 gcttcgaggg gctcgcatct ctccttcacg cgcccgccgc cctacctgag gccgccatcc
     3241 acgccggttg agtcgcgttc tgccgcctcc cgcctgtggt gcctcctgaa ctgcgtccgc
     3301 cgtctaggta agtttaaagc tcaggtcgag accgggcctt tgtccggcgc tcccttggag
     3361 cctacctaga ctcagccggc tctccacgct ttgcctgacc ctgcttgctc aactctacgt
     3421 ctttgtttcg ttttctgttc tgcgccgtta cagatccaag ctgtgaccgg cgcctactct
     3481 agagctagcg cctccggact ctagaggatc gaacccttgc caccatggac aagaagtact
     3541 ccattgggct cgatatcggc acaaacagcg tcggctgggc cgtcattacg gacgagtaca
     3601 aggtgccgag caaaaaattc aaagttctgg gcaataccga tcgccacagc ataaagaaga
     3661 acctcattgg cgccctcctg ttcgactccg gggagacggc cgaagccacg cggctcaaaa
     3721 gaacagcacg gcgcagatat acccgcagaa agaatcggat ctgctacctg caggagatct
     3781 ttagtaatga gatggctaag gtggatgact ctttcttcca taggctggag gagtcctttt
     3841 tggtggagga ggataaaaag cacgagcgcc acccaatctt tggcaatatc gtggacgagg
     3901 tggcgtacca tgaaaagtac ccaaccatat atcatctgag gaagaagctt gtagacagta
     3961 ctgataaggc tgacttgcgg ttgatctatc tcgcgctggc gcatatgatc aaatttcggg
     4021 gacacttcct catcgagggg gacctgaacc cagacaacag cgatgtcgac aaactcttta
     4081 tccaactggt tcagacttac aatcagcttt tcgaagagaa cccgatcaac gcatccggag
     4141 ttgacgccaa agcaatcctg agcgctaggc tgtccaaatc ccggcggctc gaaaacctca
     4201 tcgcacagct ccctggggag aagaagaacg gcctgtttgg taatcttatc gccctgtcac
     4261 tcgggctgac ccccaacttt aaatctaact tcgacctggc cgaagatgcc aagcttcaac
     4321 tgagcaaaga cacctacgat gatgatctcg acaatctgct ggcccagatc ggcgaccagt
     4381 acgcagacct ttttttggcg gcaaagaacc tgtcagacgc cattctgctg agtgatattc
     4441 tgcgagtgaa cacggagatc accaaagctc cgctgagcgc tagtatgatc aagcgctatg
     4501 atgagcacca ccaagacttg actttgctga aggcccttgt cagacagcaa ctgcctgaga
     4561 agtacaagga aattttcttc gatcagtcta aaaatggcta cgccggatac attgacggcg
     4621 gagcaagcca ggaggaattt tacaaattta ttaagcccat cttggaaaaa atggacggca
     4681 ccgaggagct gctggtaaag cttaacagag aagatctgtt gcgcaaacag cgcactttcg
     4741 acaatggaag catcccccac cagattcacc tgggcgaact gcacgctatc ctcaggcggc
     4801 aagaggattt ctaccccttt ttgaaagata acagggaaaa gattgagaaa atcctcacat
     4861 ttcggatacc ctactatgta ggccccctcg cccggggaaa ttccagattc gcgtggatga
     4921 ctcgcaaatc agaagagacc atcactccct ggaacttcga ggaagtcgtg gataaggggg
     4981 cctctgccca gtccttcatc gaaaggatga ctaactttga taaaaatctg cctaacgaaa
     5041 aggtgcttcc taaacactct ctgctgtacg agtacttcac agtttataac gagctcacca
     5101 aggtcaaata cgtcacagaa gggatgagaa agccagcatt cctgtctgga gagcagaaga
     5161 aagctatcgt ggacctcctc ttcaagacga accggaaagt taccgtgaaa cagctcaaag
     5221 aagactattt caaaaagatt gaatgtttcg actctgttga aatcagcgga gtggaggatc
     5281 gcttcaacgc atccctggga acgtatcacg atctcctgaa aatcattaaa gacaaggact
     5341 tcctggacaa tgaggagaac gaggacattc ttgaggacat tgtcctcacc cttacgttgt
     5401 ttgaagatag ggagatgatt gaagaacgct tgaaaactta cgctcatctc ttcgacgaca
     5461 aagtcatgaa acagctcaag aggcgccgat atacaggatg ggggcggctg tcaagaaaac
     5521 tgatcaatgg gatccgagac aagcagagtg gaaagacaat cctggatttt ctcaagtccg
     5581 atggatttgc caaccggaac ttcatgcagt tgatccatga tgactctctc acctttaagg
     5641 aggacatcca gaaagcacaa gtttctggcc agggggacag tcttcacgag cacatcgcta
     5701 atcttgcagg tagcccagct atcaaaaagg gaatactgca gaccgttaag gtcgtggatg
     5761 aactcgtcaa agtaatggga aggcataagc ccgagaatat cgttatcgag atggcccgag
     5821 agaaccaaac tacccagaag ggacagaaga acagtaggga aaggatgaag aggattgaag
     5881 agggtataaa agaactgggg tcccaaatcc tcaaggaaca cccagttgaa aacacccagc
     5941 ttcagaatga gaagctctac ctgtactacc tgcagaacgg cagggacatg tacgtggatc
     6001 aggaactgga catcaatcgg ctctccgact acgacgtgga tcatatcgtg ccccagtctt
     6061 ttctcaaaga tgattctatt gataataaag tgttgacaag atccgataaa aatagaggga
     6121 agagtgataa cgtcccctca gaagaagttg tcaagaaaat gaaaaattat tggcggcagc
     6181 tgctgaacgc caaactgatc acacaacgga agttcgataa tctgactaag gctgaacgag
     6241 gtggcctgtc tgagttggat aaagccggct tcatcaaaag gcagcttgtt gagacacgcc
     6301 agatcaccaa gcacgtggcc caaattctcg attcacgcat gaacaccaag tacgatgaaa
     6361 atgacaaact gattcgagag gtgaaagtta ttactctgaa gtctaagctg gtctcagatt
     6421 tcagaaagga ctttcagttt tataaggtga gagagatcaa caattaccac catgcgcatg
     6481 atgcctacct gaatgcagtg gtaggcactg cacttatcaa aaaatatccc aagcttgaat
     6541 ctgaatttgt ttacggagac tataaagtgt acgatgttag gaaaatgatc gcaaagtctg
     6601 agcaggaaat aggcaaggcc accgctaagt acttctttta cagcaatatt atgaattttt
     6661 tcaagaccga gattacactg gccaatggag agattcggaa gcgaccactt atcgaaacaa
     6721 acggagaaac aggagaaatc gtgtgggaca agggtaggga tttcgcgaca gtccggaagg
     6781 tcctgtccat gccgcaggtg aacatcgtta aaaagaccga agtacagacc ggaggcttct
     6841 ccaaggaaag tatcctcccg aaaaggaaca gcgacaagct gatcgcacgc aaaaaagatt
     6901 gggaccccaa gaaatacggc ggattcgatt ctcctacagt cgcttacagt gtactggttg
     6961 tggccaaagt ggagaaaggg aagtctaaaa aactcaaaag cgtcaaggaa ctgctgggca
     7021 tcacaatcat ggagcgatca agcttcgaaa aaaaccccat cgactttctc gaggcgaaag
     7081 gatataaaga ggtcaaaaaa gacctcatca ttaagcttcc caagtactct ctctttgagc
     7141 ttgaaaacgg ccggaaacga atgctcgcta gtgcgggcga gctgcagaaa ggtaacgagc
     7201 tggcactgcc ctctaaatac gttaatttct tgtatctggc cagccactat gaaaagctca
     7261 aagggtctcc cgaagataat gagcagaagc agctgttcgt ggaacaacac aaacactacc
     7321 ttgatgagat catcgagcaa ataagcgaat tctccaaaag agtgatcctc gccgacgcta
     7381 acctcgataa ggtgctttct gcttacaata agcacaggga taagcccatc agggagcagg
     7441 cagaaaacat tatccacttg tttactctga ccaacttggg cgcgcctgca gccttcaagt
     7501 acttcgacac caccatagac agaaagcggt acacctctac aaaggaggtc ctggacgcca
     7561 cactgattca tcagtcaatt acggggctct atgaaacaag aatcgacctc tctcagctcg
     7621 gtggagacag cagggctgac cccaagaaga agaggaaggt ggcggccgct gagggcagag
     7681 gaagtcttct aacatgcggt gacgtggagg agaatcccgg cccttccggg atgaccgagt
     7741 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     7801 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     7861 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     7921 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     7981 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     8041 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     8101 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     8161 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     8221 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     8281 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga atctaggtcg
     8341 acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     8401 ctccttttac gctatgtgg
//
