LOCUS       sequence-236099-        8454 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a 6X-His tagged type II-C Cas9 from H. parainfluenzae in 
            bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8454)
  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 8454)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "MBio."; 
            date: "2018-12-4"; volume: "9(6). pii: mBio.02321-18. doi"; pages: "
            10.1128/mBio.02321-18"
COMMENT     Sequence Label: pEJS1026-pMCSG7-HpaCas9
FEATURES             Location/Qualifiers
     source          1..8454
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..21
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3258..3275
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3328..3346)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3342..3389
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3513..3532)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3699..3802
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3803..3871,3872..4663)
                     /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(4021..4040)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4834..5422
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5323..5342
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5576..5593
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5608..5747
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5673..5692)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5833..5855
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5849..6040)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6849..7931)
                     /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"
     primer_bind     7893..7912
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7932..8009)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8215..8234
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     8322..8341
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8322..8340
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    8341..8365
                     /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             8380..8402
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             8413..8430
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 gagaacctgt acttccaatc caatgccatg gaaaataaaa acttacatta tatccttggt
       61 ctcgaccttg gtatcgcctc tgttggctgg gctgtggttg aaattgacga aaaagagaat
      121 cctctgcgtt taattgatgt tggtgtacgt actttcgaac gagcagaagt gcccaaaaca
      181 ggtgaaagcc ttgcgctttc tcgccgttta gcccgctccg ctcgtcgatt aactcaacgt
      241 cgcgttgctc gtctgaaaaa agcaaaacga cttctaaaat cagaaaatat cctattatcc
      301 actgatgaac gtcttcctca tcaagtttgg cagctacgtg tcgaaggtct agatcataaa
      361 ctagaacgcc aagaatgggc tgctgtgtta ttacatttaa ttaagcaccg tggttattta
      421 tctcagcgta aaaatgaaag taaaagcgaa aataaagagc taggtgcgtt attaagcggt
      481 gtagacaata atcataaatt actgcagcaa gctacatatc gctcgccagc agaacttgcc
      541 gtaaaaaaat ttgaggtcga agaaggtcat atccgtaacc aacaaggcgc ctatactcac
      601 acgtttagcc gactagactt actcgccgaa atggaactcc tcttctctcg tcaacaacac
      661 tttggtaatc cattcgcttc agaaaaatta ttagaaaatt tgaccgcact tttaatgtgg
      721 caaaaacctg ccttatctgg tgaagctatt ttaaaaatgc tcggtaaatg tacttttgaa
      781 gatgaatata aggcagctaa gaacacttat tccgcagaac gttttgtttg gataacgaaa
      841 ttaaataatt tacgtattca agaaaatggc ttagagcgtg ctttaaatga taatgaacgt
      901 ttagcattaa tggagcaacc ttatgacaaa aatagattat tctattcaca agtacgctca
      961 atattaaaat tatctgatga ggcaatcttt aaaggcctcc gttattccgg tgaggataaa
     1021 aaggccattg aaactaaagc agtactgatg gaaatgaaag cctatcacca aattcgtaaa
     1081 gtattggaag gtaataacct aaaagctgaa tgggcagaat taaaggcaaa tccaacatta
     1141 ttagatgaaa ttggtacggc attttcatta tataaaactg atgaagatat cagcgcttac
     1201 ttagcaggaa aactctctca gcctgtatta aatgccttat tggaaaatct tagttttgat
     1261 aaatttatcc aattatccct taaagcttta tataaacttc taccattaat gcaacaagga
     1321 ctacgctatg atgaggcttg tcgtgaaatt tatggcgatc attatggtaa aaaaacagaa
     1381 gaaaaccatc acttcttacc acagattcca gctgatgaaa tccgtaatcc tgtggtttta
     1441 cgaacactca cgcaagctcg taaagtgatt aatggcgtag tgagattata tggttcgcca
     1501 gctcgtattc atattgaaac gggacgagaa gttggcaaat cttacaaaga tcgtcgtgaa
     1561 ctagaaaaac gtcaggaaga aaatcgtaaa cagcgtgaaa acgcaatcaa ggaatttaaa
     1621 gagtacttcc ctcattttgc tggtgagcct aaagccaaag acattttaaa aatgcgactt
     1681 tataaacagc aaaatgcaaa atgtttatat tccggcaaac ctatcgaatt gcaccgttta
     1741 ttagaaaaag gctatgtaga agtcgatcac gccttgccgt tttcccgtac ttgggatgat
     1801 agtttcaaca ataaagtact ggtgcttgct aatgaaaatc aaaacaaagg caatttaacc
     1861 ccttttgaat ggcttgatgg taaacataac agcgaacgct ggagagcgtt caaagcatta
     1921 gttgaaacca gtgcattccc ttatgcgaaa aaacaacgca tcctaagcca aaaacttgat
     1981 gaaaagggct ttattgaacg taatttaaat gatacgcgct acgttgctcg tttcttatgt
     2041 aattttattg cagataatat gcacttaaca ggtgaaggaa aacgaaaagt atttgcttcc
     2101 aacgggcaaa ttaccgcttt acttcgtagc cgttggggat tagcaaaatc acgggaagac
     2161 aatgaccgcc atcacgcctt agatgccgtt gtggttgctt gctcaaccgt cgccatgcag
     2221 caaaaaatca cacgttttgt tcgttttgaa gctggtgatg tattcactgg tgaacgaata
     2281 gatcgtgaaa caggtgaaat tattccatta cacttcccta ctccatggca atttttcaaa
     2341 caagaagttg aaatccgaat ttttagtgat aatcctaaac tggaactaga aaatagattg
     2401 ccggatcgtc cacaagccaa tcatgaattt gtccaacctc tgtttgtatc tcgaatgcca
     2461 acacgcaaaa tgaccggtca aggacacatg gaaaccgtga aatcagcgaa acgtctcaat
     2521 gaagggataa gcgtgattaa aatgccactc actaaattaa aattaaaaga tttagaattg
     2581 atggtaaatc gtgaacgtga aaaagatctt tatgatactt taaaagctcg tctagaggct
     2641 tttaatgatg atcctgctaa agcgtttgct gaacctttta taaaaaaagg tggggctatt
     2701 gttaaatcag tacgagtaga acaaatacaa aaatctggcg tattagtccg tgaggggaat
     2761 ggtgtcgcgg ataatgcctc aatggtgaga gtggatgtat tcactaaagg tggaaaatat
     2821 ttccttgtgc caatttatac ctggcaagtc gctaagggaa ttttacctaa taaagcagca
     2881 acacaatata aagatgagga agattgggaa gtgatggata actctgcaac tttcaaattt
     2941 tcattacacc caaatgactt agtaaaatta gtcactaaaa agaaaaccat tttaggttac
     3001 tttaatggac ttaatcgtgc cacaggtaat atagatatta aagaacatga tttagataaa
     3061 tcaaaaggga aacaaggtat ttttgaaggt gttggtatta aattagccct ttccttcgaa
     3121 aaatatcaag tcgatgaact tggaaaaaat attcgtttat gtaaaccaag taaacgccaa
     3181 ccagtccgtt aaaacattgg aagtggataa cggatccgaa ttcgagctcc gtcgacaagc
     3241 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     3301 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     3361 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     3421 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3481 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3541 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3601 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacatta
     3661 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3721 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3781 aataatttga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     3841 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3901 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3961 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     4021 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     4081 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     4141 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     4201 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     4261 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     4321 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     4381 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     4441 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     4501 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     4561 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     4621 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4681 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4741 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4801 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4861 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4921 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4981 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5041 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5101 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5161 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5221 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5281 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5341 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5401 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5461 ttgctggcct tttgctcaca tgttctttct tgcgttatcc cctgattctg tggataaccg
     5521 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     5581 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     5641 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5701 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     5761 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     5821 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     5881 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     5941 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     6001 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     6061 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     6121 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     6181 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     6241 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     6301 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     6361 atgttgttgc tcaggtcgca gacgttttgc agcagcagtt gcttcacgtt cgctcgcgta
     6421 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     6481 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     6541 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     6601 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     6661 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     6721 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     6781 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     6841 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6901 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     6961 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     7021 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     7081 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     7141 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     7201 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     7261 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     7321 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     7381 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     7441 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     7501 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     7561 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     7621 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     7681 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     7741 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     7801 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     7861 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     7921 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     7981 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     8041 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     8101 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     8161 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     8221 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     8281 tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac tcactatagg
     8341 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     8401 gatatacata tgcaccatca tcatcatcat tcttctggtg tagatctggg tacc
//
