LOCUS       sequence-225063-        5676 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a 6X-His tagged type II-C anti-CRISPR protein from S. 
            muelleri in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5676)
  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 5676)
  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: pEJS664-pMCSG7-AcrIIC5Smu
FEATURES             Location/Qualifiers
     source          1..5676
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..111
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        92..110
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    111..135
                     /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             150..172
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             183..200
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             225..245
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             704..721
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(774..792)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      788..835
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(959..978)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1145..1248
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1249..1317,1318..2109)
                     /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(1467..1486)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2280..2868
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2769..2788
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3022..3039
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3054..3193
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3119..3138)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3279..3301
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3295..3486)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4295..5377)
                     /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     5339..5358
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5378..5455)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(5661..5676,1..4)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
       61 aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
      121 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata
      181 tgcaccatca tcatcatcat tcttctggtg tagatctggg taccgagaac ctgtacttcc
      241 aatccatgaa caacagcatc aagttccacg tgagctacga cggtaccgcg cgtgcgctgt
      301 ttaacaccaa ggagcaggcg gaaaaatact gcctggttga ggaaattaac gatgagatga
      361 acggctataa gcgtaaaagc tgggaggaaa agctgcgtga ggaaaactgc gcgagcgtgc
      421 aggactgggt tgagaagaac tacaccagca gctatagcga cctgttcaac atctgcgaga
      481 ttgaagtgag cagcgcgggt caactggtta agatcgacaa caccgaggtg gacgatttcg
      541 ttgaaaactg ctatggcttt accctggagg acgatctgga ggaattcaac aaggcgaaac
      601 agtacctgca aaaattttat gcggagtgcg aaaactaaaa cattggaagt ggataacgga
      661 tccgaattcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca
      721 ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
      781 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
      841 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
      901 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
      961 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1021 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1081 cttgattagg gtgatggttc acattaacgc ttacaattta ggtggcactt ttcggggaaa
     1141 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1201 gagacaataa ccctgataaa tgcttcaata atttgaaaaa ggaagagtat gagtattcaa
     1261 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     1321 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     1381 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     1441 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     1501 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     1561 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     1621 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     1681 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     1741 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg
     1801 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     1861 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     1921 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     1981 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     2041 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     2101 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     2161 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     2221 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2281 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2341 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2401 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     2461 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2521 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2581 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2641 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2701 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2761 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2821 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2881 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcttgcg
     2941 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3001 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     3061 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     3121 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     3181 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     3241 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     3301 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     3361 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     3421 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     3481 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     3541 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     3601 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     3661 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     3721 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     3781 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     3841 gcagttgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     3901 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     3961 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     4021 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     4081 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     4141 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     4201 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     4261 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4321 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4381 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     4441 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     4501 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     4561 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     4621 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     4681 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     4741 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     4801 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     4861 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4921 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4981 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     5041 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     5101 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     5161 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     5221 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     5281 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     5341 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     5401 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     5461 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     5521 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     5581 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     5641 cgagcccgat cttccccatc ggtgatgtcg gcgata
//
