LOCUS       Exported                8179 bp ds-DNA     circular SYN 04-FEB-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8179)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Feb 5, 2020 from SnapGene Viewer 5.0.7
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8179
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     regulatory      1763..1772
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1769..5014
                     /codon_start=1
                     /product="Cas9 endonuclease from the Neisseria meningitidis
                     Type II CRISPR/Cas system (Esvelt et al., 2013)"
                     /label=NmCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRV
                     FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIK
                     SLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA
                     GNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEF
                     GNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLT
                     KLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD
                     NAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRL
                     KDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT
                     EEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRK
                     EIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG
                     RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF
                     KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR
                     VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF
                     DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAE
                     KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD
                     LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK
                     TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ
                     LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE
                     GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
     CDS             5021..5041
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5048..5113
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    5268..5323
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5682..5698)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5706..5722
                     /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(5730..5760)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5775..5796
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5855..6050
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5901..6036
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6056..6190
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(6429..7017)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7188..8048)
                     /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"
     promoter        complement(8049..8153)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attctgcagt cgacggtacc
     1741 gcgggcccgg gatccaccgg tcgccaccat ggctgccttc aaacctaatt caatcaacta
     1801 catcctcggc ctcgatatcg gcatcgcatc cgtcggctgg gcgatggtag aaattgacga
     1861 agaagaaaac cccatccgcc tgattgattt gggcgtgcgc gtatttgagc gtgccgaagt
     1921 accgaaaaca ggcgactccc ttgccatggc aaggcgtttg gcgcgcagtg ttcgccgcct
     1981 gacccgccgt cgcgcccacc gcctgcttcg gacccgccgc ctattgaaac gcgaaggcgt
     2041 attacaagcc gccaattttg acgaaaacgg cttgattaaa tccttaccga atacaccatg
     2101 gcaacttcgc gcagccgcat tagaccgcaa actgacgcct ttagagtggt cggcagtctt
     2161 gttgcattta atcaaacatc gcggctattt atcgcaacgg aaaaacgagg gcgaaactgc
     2221 cgataaggag cttggcgctt tgcttaaagg cgtagccggc aatgcccatg ccttacagac
     2281 aggcgatttc cgcacaccgg ccgaattggc tttaaataaa tttgagaaag aaagcggcca
     2341 tatccgcaat cagcgcagcg attattcgca tacgttcagc cgcaaagatt tacaggcgga
     2401 gctgattttg ctgtttgaaa aacaaaaaga atttggcaat ccgcatgttt caggcggcct
     2461 taaagaaggt attgaaaccc tactgatgac gcaacgccct gccctgtccg gcgatgccgt
     2521 tcaaaaaatg ttggggcatt gcaccttcga accggcagag ccgaaagccg ctaaaaacac
     2581 ctacacagcc gaacgtttca tctggctgac caagctgaac aacctgcgta ttttagagca
     2641 aggcagcgag cggccattga ccgataccga acgcgccacg cttatggacg agccatacag
     2701 aaaatccaaa ctgacttacg cacaagcccg taagctgctg ggtttagaag ataccgcctt
     2761 tttcaaaggc ttgcgctatg gtaaagacaa tgccgaagcc tcaacattga tggaaatgaa
     2821 ggcctaccat gccatcagcc gtgcactgga aaaagaagga ttgaaagaca aaaaatcccc
     2881 attaaacctt tctcccgaat tacaagacga aatcggcacg gcattctccc tgttcaaaac
     2941 cgatgaagac attacaggcc gtctgaaaga ccgtatacag cccgaaatct tagaagcgct
     3001 gttgaaacac atcagcttcg ataagttcgt ccaaatttcc ttgaaagcat tgcgccgaat
     3061 tgtgcctcta atggaacaag gcaaacgtta cgatgaagcc tgcgccgaaa tctacggaga
     3121 ccattacggc aagaagaata cggaagaaaa gatttatctg ccgccgattc ccgccgacga
     3181 aatccgcaac cccgtcgtct tgcgcgcctt atctcaagca cgtaaggtca ttaacggcgt
     3241 ggtacgccgt tacggctccc cagctcgtat ccatattgaa actgcaaggg aagtaggtaa
     3301 atcgtttaaa gaccgcaaag aaattgagaa acgccaagaa gaaaaccgca aagaccggga
     3361 aaaagccgcc gccaaattcc gagagtattt ccccaatttt gtcggagaac ccaaatccaa
     3421 agatattctg aaactgcgcc tgtacgagca acaacacggc aaatgcctgt attcgggcaa
     3481 agaaatcaac ttaggccgtc tgaacgaaaa aggctatgtc gaaatcgacc atgccctgcc
     3541 gttctcgcgc acatgggacg acagtttcaa caataaagta ctggtattgg gcagcgaaaa
     3601 ccaaaacaaa ggcaatcaaa ccccttacga atacttcaac ggcaaagaca acagccgcga
     3661 atggcaggaa tttaaagcgc gtgtcgaaac cagccgtttc ccgcgcagta aaaaacaacg
     3721 gattctgctg caaaaattcg atgaagacgg ctttaaagaa cgcaatctga acgacacgcg
     3781 ctacgtcaac cgtttcctgt gtcaatttgt tgccgaccgt atgcggctga caggtaaagg
     3841 caagaaacgt gtctttgcat ccaacggaca aattaccaat ctgttgcgcg gcttttgggg
     3901 attgcgcaaa gtgcgtgcgg aaaacgaccg ccatcacgcc ttggacgccg tcgtcgttgc
     3961 ctgctcgacc gttgccatgc agcagaaaat tacccgtttt gtacgctata aagagatgaa
     4021 cgcgtttgac ggtaaaacca tagacaaaga aacaggagaa gtgctgcatc aaaaaacaca
     4081 cttcccacaa ccttgggaat ttttcgcaca agaagtcatg attcgcgtct tcggcaaacc
     4141 ggacggcaaa cccgaattcg aagaagccga taccctagaa aaactgcgca cgttgcttgc
     4201 cgaaaaatta tcatctcgcc ccgaagccgt acacgaatac gttacgccac tgtttgtttc
     4261 acgcgcgccc aatcggaaga tgagcgggca agggcatatg gagaccgtca aatccgccaa
     4321 acgactggac gaaggcgtca gcgtgttgcg cgtaccgctg acacagttaa aactgaaaga
     4381 cttggaaaaa atggtcaatc gggagcgcga acctaagcta tacgaagcac tgaaagcacg
     4441 gctggaagca cataaagacg atcctgccaa agcctttgcc gagccgtttt acaaatacga
     4501 taaagcaggc aaccgcaccc aacaggtaaa agccgtacgc gtagagcaag tacagaaaac
     4561 cggcgtatgg gtgcgcaacc ataacggtat tgccgacaac gcaaccatgg tgcgcgtaga
     4621 tgtgtttgag aaaggcgaca agtattatct ggtaccgatt tacagttggc aggtagcgaa
     4681 agggattttg ccggataggg ctgttgtaca aggaaaagat gaagaagatt ggcaacttat
     4741 tgatgatagt ttcaacttta aattctcatt acaccctaat gatttagtcg aggttataac
     4801 aaaaaaagct agaatgtttg gttactttgc cagctgccat cgaggcacag gtaatatcaa
     4861 tatacgcatt catgatcttg atcataaaat tggcaaaaat ggaatactgg aaggtatcgg
     4921 cgtcaaaacc gccctttcat tccaaaaata ccaaattgac gaactgggca aagaaatcag
     4981 accatgccgt ctgaaaaaac gcccgcctgt ccgtggatcc cccaagaaga agaggaaagt
     5041 ctcgagcgac tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga
     5101 tgacgatgac aagtaaagcg gccgcactcc tcaggtgcag gctgcctatc agaaggtggt
     5161 ggctggtgtg gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa
     5221 aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt
     5281 tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg
     5341 gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc
     5401 catatgctgg ctgccatgaa caaaggtggc tataaagagg tcatcagtat atgaaacagc
     5461 cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta
     5521 tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac
     5581 tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat
     5641 gaagatccct cgacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5701 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5761 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5821 ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca gcaaccatag
     5881 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     5941 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     6001 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctaactt
     6061 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     6121 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     6181 tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     6241 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     6301 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     6361 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     6421 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     6481 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     6541 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6601 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     6661 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6721 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6781 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6841 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     6901 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6961 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     7021 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     7081 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     7141 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     7201 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     7261 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     7321 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     7381 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     7441 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     7501 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     7561 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     7621 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     7681 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7741 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7801 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7861 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7921 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7981 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     8041 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     8101 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     8161 ccccgaaaag tgccacctg
//
