LOCUS       sequence-390644-        5376 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses acrIC4 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 5376)
  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 5376)
  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_acrIC4_invivo
FEATURES             Location/Qualifiers
     source          1..5376
                     /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(311..329)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      325..372
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      409..864
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(496..515)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     706..727
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(956..1771)
                     /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(956..1771)
                     /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     1679..1698
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1763..1782
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1893..2481
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2382..2401
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2635..2652
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2667..2806
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2732..2751)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2892..2914
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2908..3099)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3908..4990)
                     /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     4952..4971
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4991..5068)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5274..5293
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactatagga gaccacaacg gtttccctct agtgcctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg gataacaaaa tcacacctgc ggacgaggaa
      121 aagattcggg agtggttgaa ttgcgaagaa gcgtctgtgg ataacgacgg cgatgtgtgg
      181 gtggcagttc cgatgacggg gcattggctc agtgacgaac agaaagcaaa atatattgaa
      241 tggcgtggcg atgaaacgtg agatccggct gctaacaaag cccgaaagga agctgagttg
      301 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
      361 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
      421 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
      481 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
      541 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
      601 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
      661 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
      721 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
      781 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
      841 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
      901 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
      961 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     1021 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     1081 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     1141 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     1201 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     1261 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     1321 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     1381 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     1441 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     1501 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     1561 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     1621 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     1681 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     1741 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     1801 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     1861 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     1921 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     1981 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     2041 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2101 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2161 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2221 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     2281 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     2341 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     2401 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     2461 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     2521 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     2581 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     2641 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     2701 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     2761 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     2821 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     2881 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     2941 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     3001 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     3061 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     3121 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     3181 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     3241 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     3301 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     3361 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     3421 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     3481 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     3541 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     3601 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     3661 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     3721 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     3781 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     3841 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     3901 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     3961 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     4021 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     4081 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     4141 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     4201 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     4261 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     4321 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     4381 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     4441 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     4501 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     4561 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     4621 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     4681 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     4741 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     4801 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     4861 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     4921 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     4981 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     5041 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     5101 tgcattagga agcagcccag tagtaggttg aggccattga gcaccgccgc cgcaaggaat
     5161 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     5221 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     5281 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     5341 ccggcgtaga ggatcgagat ctcgatcccg cgaaat
//
