LOCUS       sequence-390647-        4289 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses CRISPR Cascade Type IC target sequence for in vivo assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4289)
  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 4289)
  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: pCDF_TypeIC_target_sfGFP_invivo
FEATURES             Location/Qualifiers
     source          1..4289
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..37
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        18..36
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    37..60
                     /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)."
     CDS             138..851
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             908..952
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(990..1008)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1004..1051
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1222..2013)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2014..2105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2153..2891)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(3101..4183)
                     /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     4145..4164
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4184..4261)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ctcctgcatt aggaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
       61 gccatgctca ggctggcgag tgcgccactc atcaaagata taccatgcca gcgtctcatc
      121 tttatgcgtc agttctgatg cgtaaaggcg aagagctgtt cactggtgtc gtccctattc
      181 tggtggaact ggatggtgat gtcaacggtc ataagttttc cgtgcgtggc gagggtgaag
      241 gtgacgcaac taatggtaaa ctgacgctga agttcatctg tactactggt aaactgccgg
      301 ttccttggcc gactctggta acgacgctga cttatggtgt tcagtgcttt gctcgttatc
      361 cggaccatat gaagcagcat gacttcttca agtccgccat gccggaaggc tatgtgcagg
      421 aacgcacgat ttcctttaag gatgacggca cgtacaaaac gcgtgcggaa gtgaaatttg
      481 aaggcgatac cctggtaaac cgcattgagc tgaaaggcat tgactttaaa gaggacggca
      541 atatcctggg ccataagctg gaatacaatt ttaacagcca caatgtttac atcaccgccg
      601 ataaacaaaa aaatggcatt aaagcgaatt ttaaaattcg ccacaacgtg gaggatggca
      661 gcgtgcagct ggctgatcac taccagcaaa acactccaat cggtgatggt cctgttctgc
      721 tgccagacaa tcactatctg agcacgcaaa gcgttctgtc taaagatccg aacgagaaac
      781 gcgatcatat ggttctgctg gagttcgtaa ccgcagcggg catcacgcat ggtatggatg
      841 aactgtacaa atgaggatcc aagccagtga taagtggaat gccatgtggg ctgtcctcga
      901 gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag cacatggact cgtctactag
      961 cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     1021 ggcctctaaa cgggtcttga ggggtttttt gctgaaacct caggcatttg agaagcacac
     1081 ggtcacactg cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat
     1141 ttaacgaccc tgccctgaac cgacgaccgg gtcatcgtgg ccggatcttg cggcccctcg
     1201 gcttgaacga attgttagac attatttgcc gactaccttg gtgatctcgc ctttcacgta
     1261 gtggacaaat tcttccaact gatctgcgcg cgaggccaag cgatcttctt cttgtccaag
     1321 ataagcctgt ctagcttcaa gtatgacggg ctgatactgg gccggcaggc gctccattgc
     1381 ccagtcggca gcgacatcct tcggcgcgat tttgccggtt actgcgctgt accaaatgcg
     1441 ggacaacgta agcactacat ttcgctcatc gccagcccag tcgggcggcg agttccatag
     1501 cgttaaggtt tcatttagcg cctcaaatag atcctgttca ggaaccggat caaagagttc
     1561 ctccgccgct ggacctacca aggcaacgct atgttctctt gcttttgtca gcaagatagc
     1621 cagatcaatg tcgatcgtgg ctggctcgaa gatacctgca agaatgtcat tgcgctgcca
     1681 ttctccaaat tgcagttcgc gcttagctgg ataacgccac ggaatgatgt cgtcgtgcac
     1741 aacaatggtg acttctacag cgcggagaat ctcgctctct ccaggggaag ccgaagtttc
     1801 caaaaggtcg ttgatcaaag ctcgccgcgt tgtttcatca agccttacgg tcaccgtaac
     1861 cagcaaatca atatcactgt gtggcttcag gccgccatcc actgcggagc cgtacaaatg
     1921 tacggccagc aacgtcggtt cgagatggcg ctcgatgacg ccaactacct ctgatagttg
     1981 agtcgatact tcggcgatca ccgcttccct catactcttc ctttttcaat attattgaag
     2041 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2101 acaaatagct agctcactcg gtcgctacgc tccgggcgtg agactgcggc gggcgctgcg
     2161 gacacataca aagttaccca cagattccgt ggataagcag gggactaaca tgtgaggcaa
     2221 aacagcaggg ccgcgccggt ggcgtttttc cataggctcc gccctcctgc cagagttcac
     2281 ataaacagac gcttttccgg tgcatctgtg ggagccgtga ggctcaacca tgaatctgac
     2341 agtacgggcg aaacccgaca ggacttaaag atccccaccg tttccggcgg gtcgctccct
     2401 cttgcgctct cctgttccga ccctgccgtt taccggatac ctgttccgcc tttctccctt
     2461 acgggaagtg tggcgctttc tcatagctca cacactggta tctcggctcg gtgtaggtcg
     2521 ttcgctccaa gctgggctgt aagcaagaac tccccgttca gcccgactgc tgcgccttat
     2581 ccggtaactg ttcacttgag tccaacccgg aaaagcacgg taaaacgcca ctggcagcag
     2641 ccattggtaa ctgggagttc gcagaggatt tgtttagcta aacacgcggt tgctcttgaa
     2701 gtgtgcgcca aagtccggct acactggaag gacagatttg gttgctgtgc tctgcgaaag
     2761 ccagttacca cggttaagca gttccccaac tgacttaacc ttcgatcaaa ccacctcccc
     2821 aggtggtttt ttcgtttaca gggcaaaaga ttacgcgcag aaaaaaagga tctcaagaag
     2881 atcctttgat cttttctact gaaccgctct agatttcagt gcaatttatc tcttcaaatg
     2941 tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg
     3001 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     3061 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3121 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3181 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     3241 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     3301 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     3361 cgtcgtatcc cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc
     3421 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     3481 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     3541 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     3601 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     3661 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     3721 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     3781 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     3841 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     3901 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     3961 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     4021 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     4081 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     4141 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     4201 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     4261 ccgggatctc gacgctctcc cttatgcga
//
