LOCUS       sequence-177170-        6145 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid contains the gene for anti-CRISPR protein AcrF3 (gene 
            product 35 from bacteriophage JBD5)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6145)
  AUTHORS   Rollins MF, Chowdhury S, Carter J, Golden SM, Wilkinson RA, 
            Bondy-Denomy J, Lander GC, Wiedenheft B
  TITLE     Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease 
            activity.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Apr 24. pii: 201616395. doi: 
            10.1073/pnas.1616395114.
  PUBMED    28438998
REFERENCE   2  (bases 1 to 6145)
  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: "Proc Natl 
            Acad Sci U S A. 2017 Apr 24. pii: 201616395. doi: 
            10.1073/pnas.1616395114."
COMMENT     Sequence Label: pAcrF3
FEATURES             Location/Qualifiers
     source          1..6145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             130..150
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(646..664)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      660..707
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(882..887,888..904,905..910))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(937..955)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1023..1127
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1128..1196,1197..1988)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1346..1365)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2159..2747
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2648..2667
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2901..2918
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2933..3072
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2998..3017)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3158..3180
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3174..3365)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4677..5759)
                     /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     5721..5740
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5760..5837)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6043..6062
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcagag aaaacttgta tttccagggc atgagcaaca cgatttcaga tcgcatcgtc
      181 gcccgctcgg ttattgaagc ggctcgattc atccagtcct gggaagatgc agaccccgac
      241 agcctgactg aagaccaggt cctggccgcc gctggttttg ccgccaggct ccatgagggg
      301 ctccaggcta ccgtcctgca acggctggtg gacgagtcca atcatgaaga gtatcgcgag
      361 ttcaaggcat gggaagaggc gctgctcaac gcagatgggc gggtcgcgag cagcccgttt
      421 gctgattggg gatggtggta tcgcatcgcc aacgtgatgc tggccactgc ctcgcagaac
      481 gtaggtgtca cctggggaag ccgcgtccat gggcgtttaa tggctatttt tcaggacaag
      541 ttcaagcagc ggtatgagga acaggcatga tgacgaagct tgcggccgca ctcgaggatc
      601 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
      661 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
      721 ctatatccgg atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac
      781 agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg
      841 tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct tatcgatgat
      901 aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
      961 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     1021 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1081 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     1141 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     1201 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     1261 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     1321 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     1381 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     1441 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     1501 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     1561 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     1621 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     1681 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     1741 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     1801 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     1861 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     1921 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     1981 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     2041 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     2101 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2161 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2221 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2281 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     2341 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2401 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     2461 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2521 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2581 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     2641 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     2701 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     2761 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     2821 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     2881 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     2941 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     3001 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     3061 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3121 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3181 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     3241 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     3301 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     3361 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     3421 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     3481 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     3541 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     3601 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     3661 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     3721 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     3781 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac
     3841 ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg
     3901 ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt
     3961 cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc
     4021 ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa
     4081 tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg
     4141 gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat
     4201 ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg
     4261 aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga
     4321 cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4381 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4441 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4501 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     4561 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     4621 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     4681 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4741 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     4801 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     4861 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     4921 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     4981 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     5041 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     5101 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     5161 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     5221 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5281 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5341 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5401 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     5461 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     5521 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     5581 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     5641 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     5701 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     5761 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     5821 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     5881 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     5941 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     6001 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     6061 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     6121 gatcgagatc tcgatcccgc gaaat
//
