LOCUS       Exported                8487 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses a 6X-His tagged type II-C Cas9 from S. muelleri in 
            bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    pEJS1027-pMCSG7-SmuCas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8487)
  AUTHORS   Lee J, Mir A, Edraki A, Garcia B, Amrani N, Lou HE, Gainetdinov I, 
            Pawluk A, Ibraheim R, Gao XD, Liu P, Davidson AR, Maxwell KL, 
            Sontheimer EJ
  TITLE     Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and 
            AcrIIC5 Anti-CRISPR Proteins.
  JOURNAL   MBio. 2018 Dec 4;9(6). pii: mBio.02321-18. doi: 
            10.1128/mBio.02321-18.
  PUBMED    30514786
REFERENCE   2  (bases 1 to 8487)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8487
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     824..843
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(863..940)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1146..1165
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1253..1272
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1253..1271
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1272..1296
                     /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)."
     RBS             1311..1333
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1344..1361
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1386..1406
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4676..4693
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4746..4764)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4760..4807
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4931..4950)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5117..5220
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5221..6081
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5439..5458)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6252..6840
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6741..6760
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6994..7011
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7026..7165
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7091..7110)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7251..7273
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7267..7458)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(8267..862)
                     /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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
       61 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
      121 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
      181 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
      241 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
      301 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
      361 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
      421 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
      481 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
      541 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
      601 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
      661 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
      721 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
      781 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
      841 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
      901 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
      961 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     1021 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     1081 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     1141 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     1201 gccacgatgc gtccggcgta gaggatcgag atcgatctcg atcccgcgaa attaatacga
     1261 ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac
     1321 tttaagaagg agatatacat atgcaccatc atcatcatca ttcttctggt gtagatctgg
     1381 gtaccgagaa cctgtacttc caatccaatg ccatgatgat ggaaaaattt cactatgtat
     1441 tgggtttgga tttgggtatc gcctctgtgg ggtgggctgc cattgaaatt gacaaggaaa
     1501 ccgaaacatc aatcggttta ttggattgcg gtgtcagaac atttgaacgt gcagaagtac
     1561 ccaaaacagg cgattctctt gccaaagctc gccgtgaagc cagaagtact cgccgtttaa
     1621 ttcgcagacg ttcgcatcgc ttattacgtt taaaacgttt attgaaacgt gaaattttca
     1681 ggcagcctga aacgtttaaa gacttaccaa tcaatgcttg gcaattgcgt gttaaaggct
     1741 tggatagtcg gttgaatgaa tatgaatggg cggccgtttt attgcatttg gtgaagcatc
     1801 gcggttattt atcgcaacgc aaaagcgaaa tgagcgaaac agacagcaaa tctgaaatgg
     1861 gcagattact ggcaggtgtg gcggaaaatc accaactttt acaacaagaa caatatcgta
     1921 caccagccga attagcactc aaaaaatttg tgaaacattt tcgcaataaa ggtggcgatt
     1981 atgcacacac tttcaaccgt ttggatttgc aagccgaatt gcatttattg tttcaaaaac
     2041 aacgtgaatt aggcaatcca ttcacttcac cagaattgga acggcaagtt gatgatttgt
     2101 tgatgacgca gcgcagtgct ttacaaggtg atgcgatttt gaaaatgttg ggtcattgtg
     2161 ggtttgaacc tgaacaattc aaagcagcga aaaacacatt cagtgccgaa cgttttattt
     2221 ggttgacaaa actcaataat cttcgcattc aagaccaagg caaagaacgt gcgttaactg
     2281 ccgatgagcg taccaaattg ttggacgagc cttataaaaa aagtaaattg acttacgcac
     2341 aagttcgcaa attattaagc ttgcctcaaa ctgctatttt taaaggtttg cgttatgatt
     2401 tggaacatga caaaaaagca gaaaacagta cgttgatgga aatgaaatcc tatcacaaca
     2461 tccgccaaac attggaaaaa tcaggtttga aaacagaatg gcaaagtatt gccacgcagc
     2521 ctgaaatttt agatgcaatt ggcacggcgt tttccattta taaaaccgat gaagatattt
     2581 cgcatgaatt aaaaacgtgc aggctgcctg aaaacgtatt gaatgaatta ctgaaaaaca
     2641 tcaattttga tggattcatt caattatcgt tgactgcatt acgcaaaatt ttgcccttga
     2701 tggaacaagg ctaccgttat gatgaagcgt gtacccaaat ttacggtaat catcattcag
     2761 gcagcttgca acaagaatca aagcaatttt tgccacatat tccgattgat gatgtccgaa
     2821 atcctgtggt gttccgtact ttgacccaag caagaaaagt ggtgaatgcg attattcgtc
     2881 ggtatggttc gccagctcgt gtgcatattg agatggcgcg tgaattgggt aagtctaaat
     2941 cagaccgtga ccgaattgaa aaacaacaac aaaaaaataa aaaagaacgt gaaaacgcag
     3001 tcgccaaatt caaagaagat ttccctgatt ttgtgggcga acccagaggg aaagatattt
     3061 tgaaaatgcg tttgtatgaa caacaacacg gcaaatgttt gtattcgggt catgatattg
     3121 atattaatcg attgaatgaa aaaggttatg ttgaaattga ccatgccctg ccattttcac
     3181 ggacttggga tgatagtcaa aataataaag ttttggtact tggcagcgaa aaccaaaata
     3241 aacgcaatca aacgcctgat gaatatttgg acggtgcaaa caatagccaa cgttggcttg
     3301 aatttcaagc gcgtgtacaa acttgtcatt tttcttacgg taaaaaacaa cgcattcaat
     3361 tagccaaatt agacgatgaa accgaaaaag gatttttaga acgcaatcta aatgatacgc
     3421 ggtatattgc tcgttttatg tgtcaatttg tccaagaaaa tttatatttg acaggtaaag
     3481 gaaaacgtct tgtttttgca tcaaacggcg gaatgaccgc aacattgaga aatttatggg
     3541 gtttgagaaa agtccgtgaa gacaacgacc gccatcatgc tcttgatgcg attgtggtgg
     3601 cgtgttccac tgcttctatg caacaaaaaa taaccaaagc atttcaacgc catgaaagca
     3661 ttgaatatgt ggataccgaa acgggcgaag taaaatttcg tattccacag ccttgggatt
     3721 ttttccgtca ggaagtgatg attcgtgtgt tcagcgacca accgtgtgaa gatttggtag
     3781 aaaaattgtc ggctcgtccc gaagctttgc atgacaacgt aacgccttta tttgtctcgc
     3841 gtgcaccaaa tcgcaaaatg tcggggcaag ggcatttgga aaccatcaaa tctgcaaaaa
     3901 ggctgtctga agaaaacagt atggttaaaa aaccattaac cacattgaaa ttaaaagata
     3961 ttccagaaat cgtaggctac ccgagtcgtg aacctcaatt gtatgccgca ttgaaaacac
     4021 gtttagaaac gcatgatgat gacccaatta aagcctttgc caaacccttt tacaaaccca
     4081 ataaaaatgg tgaattgggg gcgttggttc gatcggtgcg tgtgaaaggt gtacaaaata
     4141 cgggtgtaat ggttcatgat ggcaaaggca ttgccgataa tgccacaatg gttcgtgttg
     4201 atgtctatac caaagcgggc aaaaattacc ttgttcctgt gtatgtttgg caggtggctc
     4261 aaggaatttt gccaaatcgg gcggttactt ctggcaaaag tgaagcagat tgggatttaa
     4321 ttgatgaaag ttttgaattt aaattttcgc tgtctcgtgg ggatttagtg gaaatgatta
     4381 gcaataaagg aagaattttt ggttattaca atgggttaga tcgtgcaaat ggaagtattg
     4441 ggattcgtga acatgatttg gaaaagtcca aaggaaaaga tggtgttcat cgtgttggcg
     4501 tgaaaaccgc caccgcattc aacaaatacc acgttgaccc acttggtaaa gaaattcatc
     4561 ggtgttcatc tgaaccacgc cccacattaa aaatcaaatc caagaaataa aacattggaa
     4621 gtggataacg gatccgaatt cgagctccgt cgacaagctt gcggccgcac tcgagcacca
     4681 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     4741 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     4801 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     4861 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4921 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4981 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5041 gaccccaaaa aacttgatta gggtgatggt tcacattaac gcttacaatt taggtggcac
     5101 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5161 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatttgaaa aaggaagagt
     5221 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5281 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5341 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5401 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5461 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5521 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5581 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5641 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5701 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5761 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5821 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5881 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5941 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6001 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6061 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6121 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6181 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6241 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6301 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6361 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     6421 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6481 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6541 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6601 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6661 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6721 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6781 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6841 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6901 ttctttcttg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     6961 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     7021 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     7081 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     7141 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     7201 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     7261 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     7321 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     7381 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     7441 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     7501 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     7561 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     7621 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     7681 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     7741 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     7801 cgttttgcag cagcagttgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     7861 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     7921 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     7981 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     8041 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     8101 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     8161 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     8221 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     8281 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     8341 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     8401 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     8461 ccagcaggcg aaaatcctgt ttgatgg
//
