LOCUS       Exported                8548 bp ds-DNA     circular SYN 06-JUL-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Untitled
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8548)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Saturday, July 6, 2019 from SnapGene Viewer 3.1.2
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8548
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(185..773)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(944..1804)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(1805..1909)
                     /gene="bla"
                     /note="AmpR promoter"
     promoter        1962..2202
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        2232..2307
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        2465..2844
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2845..3048
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     promoter        3093..3111
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3151..3216
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /note="3xFLAG"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3223..3243
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             3268..7368
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /note="Cas9"
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7369..7416
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /note="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7432..7488
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /note="P2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7495..8094
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /note="PuroR"
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    8332..8379
                     /note="HSV TK poly(A) signal"
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
       61 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
      121 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
      181 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
      241 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
      301 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
      361 gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc
      421 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
      481 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
      541 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
      601 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
      661 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
      721 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
      781 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
      841 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
      901 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
      961 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     1021 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     1081 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     1141 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     1201 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     1261 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     1321 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     1381 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     1441 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     1501 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     1561 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     1621 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     1681 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     1741 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     1801 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     1861 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     1921 gaaaagtgcc acctgacggt accgctagcg tcgacggatc cgagggccta tttcccatga
     1981 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
     2041 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     2101 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     2161 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gaccccacag
     2221 tggggccact agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
     2281 cttgaaaaag tggcaccgag tcggtgcttt ttttagatct cgagtctaga ggaggtcgct
     2341 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
     2401 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
     2461 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     2521 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     2581 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     2641 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     2701 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     2761 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     2821 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     2881 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     2941 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     3001 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
     3061 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
     3121 gctgcccggg atccaccggt cgccaccatg gactataagg accacgacgg agactacaag
     3181 gatcatgata ttgattacaa agacgatgac gataagatgg ccccaaagaa gaagcggaag
     3241 gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct ggacatcggc
     3301 accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc
     3361 aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg
     3421 ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac
     3481 accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag
     3541 gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag
     3601 cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac
     3661 cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg
     3721 ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc
     3781 gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac
     3841 aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg
     3901 tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag
     3961 aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc
     4021 aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac
     4081 gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc
     4141 gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc
     4201 accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg
     4261 accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc
     4321 gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc
     4381 tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag
     4441 ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac
     4501 cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc
     4561 ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg
     4621 ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc
     4681 atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc
     4741 gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc
     4801 ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag
     4861 ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg
     4921 ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc
     4981 gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc
     5041 acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac
     5101 gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc
     5161 gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag
     5221 cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac
     5281 aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac
     5341 ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag
     5401 gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc
     5461 attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc
     5521 cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag
     5581 ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc
     5641 agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac
     5701 ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg
     5761 ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga cgactccatc
     5821 gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc
     5881 gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt
     5941 acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat
     6001 aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca
     6061 cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa
     6121 gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt
     6181 tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc
     6241 gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac
     6301 tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct
     6361 accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg
     6421 gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc
     6481 gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg
     6541 aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc
     6601 aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc
     6661 ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc
     6721 aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc
     6781 agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag
     6841 gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga
     6901 atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat
     6961 gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat
     7021 gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag
     7081 atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc
     7141 gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg
     7201 tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac
     7261 cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc
     7321 accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa aaggccggcg
     7381 gccacgaaaa aggccggcca ggcaaaaaag aaaaaggaat tcggaagcgg cgcaacaaac
     7441 ttctcactac tcaaacaagc aggtgacgtg gaggagaatc ccgggcctct taagatgacc
     7501 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     7561 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     7621 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     7681 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     7741 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     7801 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     7861 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     7921 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     7981 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     8041 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaatcgat
     8101 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     8161 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     8221 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     8281 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     8341 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     8401 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     8461 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     8521 tcggggcggc aggccctgcc atagcctc
//
