LOCUS       Exported                7885 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 7885)
  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..7885
                     /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..4720
                     /codon_start=1
                     /product="Cas9 endonuclease from the Campylobacter jejuni 
                     Type II CRISPR/Cas system (Kim et al., 2016)"
                     /label=CjCas9
                     /translation="MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLA
                     LPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYEL
                     RFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVG
                     EYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKF
                     EEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNT
                     EGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIK
                     ALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFK
                     ALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKE
                     YRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGL
                     KINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVL
                     VFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDR
                     NLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTW
                     GFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKR
                     KFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKAL
                     ELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGE
                     IKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFET
                     LSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK"
     CDS             4727..4747
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4754..4819
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    4974..5029
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5388..5404)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5412..5428
                     /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(5436..5466)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5481..5502
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5561..5756
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5607..5742
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5762..5896
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(6135..6723)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6894..7754)
                     /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(7755..7859)
                     /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 ggccagaatc ctggccttcg acatcggcat
     1801 cagcagcatc ggctgggcct tcagcgagaa cgacgagctg aaggactgcg gcgtgcggat
     1861 cttcaccaag gtggaaaacc ccaagaccgg cgagagcctg gccctgccca gaaggctggc
     1921 cagaagcgcc cggaagagac tggccagacg gaaggcccgg ctgaaccacc tgaagcacct
     1981 gatcgccaac gagttcaagc tgaactacga ggactaccag agcttcgacg agtccctggc
     2041 caaggcctac aagggcagcc tgatcagccc ctacgagctg cggttccggg ccctgaacga
     2101 gctgctgagc aagcaggact tcgccagagt gatcctgcac attgccaagc ggagaggcta
     2161 cgacgacatc aagaacagcg acgacaaaga gaagggcgcc atcctgaagg ccatcaagca
     2221 gaacgaggaa aagctggcca actaccagtc cgtgggcgag tacctgtaca aagagtactt
     2281 ccagaagttc aaagagaaca gcaaagaatt caccaacgtg cggaacaaga aagaaagcta
     2341 cgagcggtgt atcgcccaga gcttcctgaa ggatgagctg aagctgatct tcaagaagca
     2401 gagagagttc ggcttcagct tcagcaagaa attcgaggaa gaggtgctga gcgtcgcctt
     2461 ctacaagaga gccctgaagg acttcagcca cctcgtgggc aactgcagct tcttcaccga
     2521 cgagaagaga gcccccaaga acagccccct ggccttcatg ttcgtggccc tgacccggat
     2581 catcaacctg ctgaacaatc tgaagaacac cgagggcatc ctgtacacca aggacgacct
     2641 gaacgccctg ctgaatgagg tgctgaagaa cggcaccctg acctacaagc agaccaagaa
     2701 gctgctgggc ctgagcgacg actacgagtt taagggcgag aagggcacct acttcatcga
     2761 gttcaagaag tacaaagagt tcatcaaggc cctgggcgag cacaacctga gccaggacga
     2821 tctgaatgag atcgccaagg acatcaccct gatcaaggac gagattaagc tgaagaaggc
     2881 cctggccaaa tacgacctga atcagaacca gatcgacagc ctgagcaagc tggaattcaa
     2941 ggatcacctg aacatcagct tcaaggctct gaagctggtc acccccctga tgctggaagg
     3001 caagaagtac gacgaggcct gcaacgagct gaacctgaag gtggccatca acgaggacaa
     3061 gaaggacttc ctgcccgcct tcaacgaaac ctactacaag gacgaagtga ccaaccccgt
     3121 ggtgctgcgg gccatcaaag aataccggaa ggtgctgaat gccctgctca agaaatacgg
     3181 caaggtgcac aagatcaaca tcgagctggc ccgggaagtg ggcaagaacc acagccagcg
     3241 ggccaagatc gagaaagagc agaacgaaaa ctacaaggcc aagaaggacg ctgagctgga
     3301 atgcgagaag ctgggactga agatcaacag caagaacatc ctgaagctgc ggctgttcaa
     3361 agaacagaaa gagttctgcg cctacagcgg cgagaagatc aagatcagcg atctgcagga
     3421 cgagaagatg ctggaaatcg accacatcta cccctacagc cggtccttcg acgacagcta
     3481 catgaacaag gtgctggtgt tcaccaaaca gaaccaggaa aaactgaacc agaccccctt
     3541 cgaggccttc ggcaacgaca gcgccaagtg gcagaaaatc gaggtgctgg ccaagaacct
     3601 gcccaccaag aaacagaaga gaatcctgga caagaattac aaggacaaag agcagaagaa
     3661 cttcaaggac cggaacctga acgacacccg gtatatcgcc cggctggtgc tgaactacac
     3721 aaaggactac ctggatttcc tgcccctgtc cgacgacgag aacaccaagc tgaacgatac
     3781 ccagaaaggc tccaaggtgc acgtggaagc caagagcggc atgctgacca gcgccctgag
     3841 acacacctgg ggcttcagcg ccaaggatcg gaacaaccat ctgcaccacg ccatcgacgc
     3901 cgtgatcatt gcctacgcca acaacagcat cgtgaaggcc ttctccgact tcaagaaaga
     3961 acaggaaagc aacagcgccg agctgtacgc caagaagatc tctgagctgg actacaagaa
     4021 caagcggaag ttcttcgagc ccttcagcgg cttccggcag aaggtgctgg ataagatcga
     4081 cgagatcttc gtgtccaagc ccgagcggaa gaagccctct ggcgccctgc acgaggaaac
     4141 cttcagaaaa gaggaagagt tctaccagtc ctacggcggc aaagaaggcg tgctgaaggc
     4201 cctcgagctg ggcaagatca gaaaagtgaa cggcaagatc gtgaagaacg gggacatgtt
     4261 ccgggtggac atcttcaagc acaaaaagac caacaagttc tacgccgtgc ccatctacac
     4321 catggacttc gccctgaagg tgctgcccaa caaggccgtg gcccggtcca agaagggcga
     4381 gatcaaggac tggattctga tggacgagaa ctacgagttc tgctttagcc tgtacaagga
     4441 ctccctgatc ctgatccaga ccaaggacat gcaggaaccc gagttcgtct actacaacgc
     4501 cttcaccagc agcaccgtgt ccctgatcgt gtctaagcac gacaacaagt tcgagacact
     4561 gagcaagaac cagaagatcc tgttcaagaa cgccaacgag aaagaagtga tcgccaagag
     4621 catcggcatc cagaatctga aggtgttcga gaagtacatc gtgtccgccc tgggagaagt
     4681 gacaaaggcc gagttccggc agagagagga cttcaaaaag ggatccccca agaagaagag
     4741 gaaagtctcg agcgactaca aagaccatga cggtgattat aaagatcatg acatcgatta
     4801 caaggatgac gatgacaagt aaagcggccg cactcctcag gtgcaggctg cctatcagaa
     4861 ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc tttttccctc
     4921 tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag
     4981 gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac
     5041 atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac
     5101 atatgccata tgctggctgc catgaacaaa ggtggctata aagaggtcat cagtatatga
     5161 aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt
     5221 tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg
     5281 ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc
     5341 tcttatgaag atccctcgac ctgcagccca agcttggcgt aatcatggtc atagctgttt
     5401 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5461 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5521 cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa
     5581 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     5641 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     5701 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     5761 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     5821 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5881 ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5941 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     6001 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     6061 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     6121 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     6181 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     6241 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     6301 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     6361 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6421 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6481 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6541 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     6601 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6661 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6721 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6781 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6841 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6901 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6961 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     7021 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     7081 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     7141 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     7201 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     7261 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     7321 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     7381 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7441 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7501 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7561 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7621 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7681 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7741 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7801 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7861 acatttcccc gaaaagtgcc acctg
//
