LOCUS       Exported                8302 bp ds-DNA     circular SYN 03-JUN-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    pMLIB-CjCas9 (WT)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8302)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Jun 3, 2021 from SnapGene 4.3.11
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8302
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /note="color: #ffffff"
     rep_origin      12..467
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     CDS             complement(560..1375)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /note="color: #993366"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      1497..2085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     misc_feature    2271..2413
                     /label=bom
                     /note="basis of mobility region from pBR322"
                     /note="color: #a6acb3"
     CDS             complement(2515..2706)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /note="color: #993366"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3515..4597)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /note="color: #993366"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(4598..4675)
                     /gene="lacI"
                     /label=lacI promoter
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     4884..4900
                     /label=Primer 1
                     /note="color: black; sequence: gatgtcggcgatatagg; added: 
                     2021-06-03"
     promoter        4984..5002
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff"
     protein_bind    5003..5027
                     /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)."
                     /note="color: #31849b"
     RBS             5042..5064
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
                     /note="color: #a6acb3"
     CDS             5083..5100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #993366"
                     /translation="HHHHHH"
     CDS             5101..5121
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /note="color: #993366"
                     /translation="ENLYFQS"
     CDS             5128..8076
                     /codon_start=1
                     /product="Cas9 endonuclease from the Campylobacter jejuni 
                     Type II CRISPR/Cas system (Kim et al., 2016)"
                     /label=CjeCas9
                     /label=CjCas9
                     /note="color: #993366"
                     /translation="ARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLAL
                     PRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELR
                     FRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGE
                     YLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFE
                     EEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTE
                     GILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKA
                     LGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKA
                     LKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEY
                     RKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLK
                     INSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLV
                     FTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRN
                     LNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWG
                     FSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRK
                     FFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALE
                     LGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEI
                     KDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETL
                     SKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK"
     misc_feature    5146..5148
                     /label=RuvC D8
                     /note="color: #a6acb3"
     misc_feature    6799..6801
                     /label=HNH H559
                     /note="color: #a6acb3"
     CDS             8146..8163
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #993366"
                     /translation="HHHHHH"
     terminator      8230..8277
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
                     /note="color: #ffffff"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaact agtaacgttt acaatttcag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
      601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
      661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
      721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
      781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
      841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
      901 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
      961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatgt
     1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
     2341 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     2401 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2461 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     2521 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     2581 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     2641 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     2701 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     2761 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     2821 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     2881 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     2941 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     3001 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     3061 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     3121 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     3181 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     3241 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     3301 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     3361 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     3421 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     3481 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3541 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3601 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     3661 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     3721 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     3781 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     3841 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     3901 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     3961 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     4021 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     4081 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4141 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4201 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     4261 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     4321 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     4381 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     4441 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     4501 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     4561 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     4621 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     4681 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     4741 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     4801 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     4861 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     4921 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     4981 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     5041 ttttgtttaa ctttaagaag gagatatacc atgggttctt ctcaccatca ccatcaccat
     5101 gaaaacctgt acttccaatc caatgcagca agaattttgg catttgatat aggaataagc
     5161 tctattggct gggcatttag tgaaaatgat gagttaaaag attgtggagt taggattttt
     5221 acaaaggttg aaaatcccaa aacaggagaa tccttagcct tgccaagacg ccttgcaaga
     5281 agtgctagaa aacgccttgc tagacgcaaa gcaaggctaa atcacttaaa acatttaatc
     5341 gcaaatgaat ttaagttaaa ttacgaagat tatcaaagct ttgatgaaag tttagccaag
     5401 gcttataaag gatctttaat tagcccttat gagttaagat ttagagcttt aaacgagctt
     5461 ttaagtaagc aagattttgc aagagtgatt ttacatattg caaaaaggcg tggctatgat
     5521 gatattaaaa atagtgatga taaagaaaaa ggtgcgattt taaaagctat caagcaaaat
     5581 gaagaaaaac tagcaaatta tcaaagtgtg ggcgagtatc tttataaaga gtattttcaa
     5641 aaattcaaag aaaattccaa agaatttaca aatgttagaa ataaaaaaga aagttatgaa
     5701 cgctgcatag cgcaaagttt tttaaaagat gaattaaagc ttatttttaa aaaacaaaga
     5761 gaatttggct tttctttttc aaaaaaattt gaagaagagg tgcttagtgt agcattttat
     5821 aaaagggctt taaaagattt ttctcatttg gtgggaaatt gtagtttttt taccgatgaa
     5881 aaaagagcgc caaaaaattc gcccttagct tttatgtttg tagcacttac aagaattatc
     5941 aatcttttaa acaatcttaa aaatactgaa ggaattttat atacaaaaga tgatttaaac
     6001 gcccttttaa atgaagtttt aaaaaacggc actctaactt ataaacaaac aaaaaagctt
     6061 ttgggcttaa gcgatgatta tgaatttaaa ggagaaaaag gaacttattt tattgagttt
     6121 aaaaaataca aagaatttat aaaagcccta ggtgagcata atttaagcca agatgattta
     6181 aatgaaatag caaaagatat tactcttata aaagatgaga ttaaacttaa aaaggcttta
     6241 gcaaaatatg atttaaatca aaatcaaatt gatagtttaa gcaaattgga atttaaagac
     6301 catttaaata taagttttaa agccttaaaa cttgtcactc ctttaatgct tgaaggtaaa
     6361 aaatacgatg aagcttgcaa tgagcttaat ttaaaagtag caatcaatga agataaaaaa
     6421 gactttttac ccgcatttaa tgaaacttat tacaaagatg aagtaactaa tcctgtggtt
     6481 ttaagggcga ttaaagaata tagaaaggtc ttaaatgcct tacttaaaaa atacggtaaa
     6541 gtgcataaaa taaacattga attagcaaga gaagtaggta aaaatcacag ccaaagagct
     6601 aaaatagaaa aagagcaaaa cgaaaactac aaggccaaaa aagacgccga acttgaatgt
     6661 gaaaaactag gcttaaaaat aaatagcaaa aatattttaa aattaagatt atttaaagaa
     6721 caaaaagaat tttgtgctta tagtggagaa aagattaaaa ttagtgattt gcaagatgaa
     6781 aaaatgcttg aaattgatca catttaccct tattctagaa gttttgatga ttcttatatg
     6841 aataaagttt tagtttttac caagcaaaat caagaaaaac ttaatcaaac cccctttgaa
     6901 gcctttggca atgatagtgc aaaatggcag aaaattgaag ttttagcaaa aaatttgcct
     6961 actaaaaagc aaaaaagaat tttagataaa aattacaaag acaaagagca aaaaaacttt
     7021 aaagatagaa atctaaacga tactcgctac atagcaaggc ttgttttaaa ctacactaaa
     7081 gattatttag attttttacc tttaagcgat gatgaaaata caaaattaaa cgatacacaa
     7141 aaaggctcaa aagtgcatgt agaagcaaaa agcggtatgc ttacatcggc tttaagacac
     7201 acttggggct ttagcgctaa ggatagaaat aaccatcttc atcacgccat cgatgcagta
     7261 attattgctt atgcaaacaa ctctatcgta aaggcttttt cagattttaa aaaagagcaa
     7321 gaaagcaaca gtgcagaact ttatgcaaaa aaaattagtg aattagatta taaaaacaaa
     7381 aggaagtttt ttgagccttt tagtggtttt aggcaaaagg ttttagataa aattgatgaa
     7441 atttttgtct caaaacctga gagaaaaaag ccaagtggag ctttacatga agaaacattt
     7501 agaaaagaag aagaattcta tcaatcatac ggcggaaaag aaggagtttt aaaagcccta
     7561 gagcttggta aaatccgcaa agtaaatggt aaaatagtta aaaatggcga tatgtttaga
     7621 gtggatattt ttaaacacaa aaagacaaat aaattttatg ctgtgccaat ttatactatg
     7681 gattttgctt taaaagtatt accaaataaa gctgtagcac gaagtaaaaa aggtgagata
     7741 aaagactgga ttttaatgga tgaaaattat gagttttgct tttctttata taaagatagt
     7801 ttgattttaa ttcaaactaa agatatgcaa gagccagaat ttgtttatta taatgctttt
     7861 acaagctcta ctgttagttt gattgtttca aaacacgata ataaatttga aacgctaagc
     7921 aaaaatcaaa aaatattgtt taaaaatgca aatgaaaaag aagtaatcgc aaaaagcata
     7981 ggaattcaaa atctaaaagt atttgaaaaa tacatagttt cagcacttgg ggaagttaca
     8041 aaggctgagt ttagacaaag agaagatttt aaaaaataat aacattggaa gtggataacg
     8101 gatccgaatt cgagcgccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac
     8161 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     8221 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     8281 aaaggaggaa ctatatccgg at
//
