LOCUS       sequence-390621-        5478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses acrIF2 protein targeting CRISPR Cascade Type IC for in 
            vivo assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5478)
  AUTHORS   O'Brien RE, Bravo JPK, Ramos D, Hibshman GN, Wright JT, Taylor DW
  TITLE     Structural snapshots of R-loop formation by a type I-C CRISPR 
            Cascade.
  JOURNAL   Mol Cell. 2023 Mar 2;83(5):746-758.e5. doi: 
            10.1016/j.molcel.2023.01.024. Epub 2023 Feb 16.
  PUBMED    36805026
REFERENCE   2  (bases 1 to 5478)
  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"; doi: 
            "10.1016/j.molcel.2023.01.024"; journalName: "Mol Cell"; date: 
            "2023-03-2- 2"; volume: "83"; issue: "5"; pages: "746-758.e5"
COMMENT     Sequence Label: pET28b_TypeIC_acrIF2_invivo
FEATURES             Location/Qualifiers
     source          1..5478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(413..431)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      427..474
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      511..966
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(598..617)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     808..829
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1058..1873)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1058..1873)
                     /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"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1781..1800
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1865..1884
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1995..2583
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2484..2503
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2737..2754
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2769..2908
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2834..2853)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2994..3016
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3010..3201)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4010..5092)
                     /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     5054..5073
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5093..5170)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5376..5395
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactatagga gaccacaacg gtttccctct agtgcctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg atgatcgcgc aacagcataa ggatactgta
      121 gctgcgtgcg aggccgccga agcaatcgcc atcgctaaag accaagtctg ggatggtgag
      181 ggttatacta aatacacatt cgacgacaac tcggtgttaa tccaatccgg tactacccag
      241 tacgcaatgg atgctgatga cgcggactct atcaagggct acgcagattg gctcgacgat
      301 gaggcgcggt cggcggaggc aagtgaaatt gaacgcctgc tggagtccgt agaggaggag
      361 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
      421 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
      481 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
      541 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
      601 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
      661 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
      721 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
      781 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
      841 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
      901 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
      961 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1021 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     1081 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     1141 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     1201 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     1261 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     1321 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     1381 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     1441 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     1501 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     1561 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     1621 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     1681 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     1741 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     1801 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     1861 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     1921 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     1981 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     2041 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     2101 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     2161 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     2221 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     2281 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     2341 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     2401 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     2461 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     2521 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     2581 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     2641 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     2701 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     2761 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     2821 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     2881 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     2941 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3001 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     3061 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     3121 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     3181 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     3241 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     3301 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     3361 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     3421 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     3481 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     3541 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     3601 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     3661 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     3721 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     3781 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     3841 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     3901 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     3961 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     4021 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4081 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     4141 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     4201 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     4261 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     4321 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     4381 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     4441 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     4501 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     4561 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     4621 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     4681 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     4741 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     4801 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     4861 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     4921 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     4981 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     5041 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     5101 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     5161 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     5221 agtagtaggt tgaggccatt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     5281 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     5341 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     5401 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     5461 atctcgatcc cgcgaaat
//
