LOCUS       sequence-296090-        8762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For CRISPRi. Arabinose-inducible ddcpf1 gene with CRISPR array 
            targeting the mrfp reporter gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8762)
  AUTHORS   Jervis AJ, Hanko EKR, Dunstan MS, Robinson CJ, Takano E, Scrutton NS
  TITLE     A plasmid toolset for CRISPR-mediated genome editing and CRISPRi 
            gene regulation in Escherichia coli.
  JOURNAL   Microb Biotechnol. 2021 Mar 12. doi: 10.1111/1751-7915.13780.
  PUBMED    33710766
REFERENCE   2  (bases 1 to 8762)
  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: "Microb 
            Biotechnol. 2021 Mar 12. doi: 10.1111/1751-7915.13780."
COMMENT     Sequence Label: pBbS8c-ddcpf1-rfp
FEATURES             Location/Qualifiers
     source          1..8762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(7..885)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        912..1196
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1119..1138
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     regulatory      4952..4961
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        join(5341..5346,5347..5363,5364..5370)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5378..5394
                     /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)."
     terminator      5505..5576
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5592..5619
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(6150..7100)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     terminator      complement(7862..7956)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7977..8636)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     8551..8570
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(8637..8739)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gacgtcttat gacaacttga cggctacatc attcactttt tcttcacaac cggcacggaa
       61 ctcgctcggg ctggccccgg tgcatttttt aaatacccgc gagaaataga gttgatcgtc
      121 aaaaccaaca ttgcgaccga cggtggcgat aggcatccgg gtggtgctca aaagcagctt
      181 cgcctggctg atacgttggt cctcgcgcca gcttaagacg ctaatcccta actgctggcg
      241 gaaaagatgt gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgc tggcgatatc
      301 aaaattgctg tctgccaggt gatcgctgat gtactgacaa gcctcgcgta cccgattatc
      361 catcggtgga tggagcgact cgttaatcgc ttccatgcgc cgcagtaaca attgctcaag
      421 cagatttatc gccagcagct ccgaatagcg cccttcccct tgcccggcgt taatgatttg
      481 cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga accccgtatt
      541 ggcaaatatt gacggccagt taagccattc atgccagtag gcgcgcggac gaaagtaaac
      601 ccactggtga taccattcgc gagcctccgg atgacgaccg tagtgatgaa tctctcctgg
      661 cgggaacagc aaaatatcac ccggtcggca aacaaattct cgtccctgat ttttcaccac
      721 cccctgaccg cgaatggtga gattgagaat ataacctttc attcccagcg gtcggtcgat
      781 aaaaaaatcg agataaccgt tggcctcaat cggcgttaaa cccgccacca gatgggcatt
      841 aaacgagtat cccggcagca ggggatcatt ttgcgcttca gccatacttt tcatactccc
      901 gccattcaga gaagaaacca attgtccata ttgcatcaga cattgccgtc actgcgtctt
      961 ttactggctc ttctcgctaa ccaaaccggt aaccccgctt attaaaagca ttctgtaaca
     1021 aagcgggacc aaagccatga caaaaacgcg taacaaaagt gtctataatc acggcagaaa
     1081 agtccacatt gattatttgc acggcgtcac actttgctat gccatagcat ttttatccat
     1141 aagattagcg gattctacct gacgcttttt atcgcaactc tctactgttt ctccataccc
     1201 gtttttttgg gaattcaaaa gatctacacc gtcataaaat gcctcgcatg tcagtgttat
     1261 tctcttgcgc ggttcataat ccatgaaact gtttttatgt aggaggtcgt gatgacccag
     1321 tttgaaggtt ttaccaatct ttatcaggta agcaagacgc tgagatttga gctgattccc
     1381 cagggcaaaa cactgaaaca cattcaagag caaggcttca ttgaagagga taaggcccga
     1441 aacgaccatt ataaagaact taaaccgatt atcgatcgca tctacaagac gtatgcagac
     1501 cagtgtctgc aactggtgca gttagattgg gaaaatttaa gcgcggctat agactcttac
     1561 cgtaaggaga aaacagaaga aactcgtaat gcattaatcg aggagcaggc tacataccgc
     1621 aatgcaattc atgactattt tattggtagg acagataatc taacggacgc catcaacaag
     1681 cgtcatgccg aaatatacaa aggtctgttt aaagcggagc tgttcaacgg taaagtatta
     1741 aagcagttag gtaccgtaac gaccacggaa catgaaaatg ccttgctgcg ttcttttgat
     1801 aaattcacta cgtatttttc cgggttctac gagaatcgca agaacgtgtt tagcgcagaa
     1861 gatattagta cggccattcc gcatcgtatt gttcaagaca attttccgaa atttaaagaa
     1921 aattgtcata tttttactcg tctgatcaca gcagtgccat cactgcgaga acattttgaa
     1981 aatgttaaga aggcgattgg cattttcgtg tcaaccagca ttgaagaagt gttttcattc
     2041 ccgttctata atcagcttct tacccagaca cagattgacc tatataatca gctcttaggt
     2101 ggtattagcc gagaagctgg aacggaaaag ataaaaggcc tgaatgaggt gctgaacctg
     2161 gcaattcaaa agaacgatga aacagcgcac attattgcat ctctccccca tcgatttata
     2221 ccgctcttta aacaaattct gtccgaccgc aacacactga gctttatcct ggaggaattt
     2281 aaaagcgatg aagaagtaat tcagagcttc tgcaaatata aaacgttact gcgcaatgaa
     2341 aacgtcctgg aaacggctga ggccctcttt aacgaactga acagcattga tctcacccat
     2401 atttttattt cacataaaaa gctggaaact atcagcagcg cgctgtgcga ccattgggac
     2461 acgctgagaa atgccctgta tgaacgtcga atttccgagc tgaccggaaa gatcaccaag
     2521 tcagctaaag agaaagtcca gcgctccctc aaacatgaag atattaactt acaagagatt
     2581 atttcggcgg ccggtaagga actgtcggag gctttcaaac agaagacaag cgaaatcctc
     2641 tcacatgcgc acgcggccct agatcagcct ctgccgacga cgctgaaaaa gcaggaagag
     2701 aaagaaatcc tgaaatctca attagattct ctactgggac tgtaccactt gctggactgg
     2761 tttgcggtgg acgagtctaa tgaggtggac cctgaattta gcgcacgcct gaccggtata
     2821 aaattggaga tggaaccgtc gttgagcttt tacaacaaag cacgcaacta tgcgaccaaa
     2881 aagccttatt cagtggaaaa attcaaattg aacttccaaa tgccgaccct agcgtcaggt
     2941 tgggatgtca acaaagagaa gaacaacggt gcgatcttat ttgtgaaaaa cggcctgtat
     3001 tatctgggca ttatgccgaa gcagaaaggc cgttacaagg cactgtcatt cgaaccgacg
     3061 gagaagacca gtgaagggtt tgataaaatg tattacgact actttccgga tgccgccaaa
     3121 atgattccga agtgttcgac ccagctgaaa gcggtgaccg cgcattttca gacacacacc
     3181 accccgatcc tgctttcaaa taactttata gaaccgctgg aaataaccaa agaaatttac
     3241 gacctgaaca atccggaaaa agaaccgaag aaatttcaga ccgcctacgc aaagaaaacc
     3301 ggagatcaaa aaggttaccg cgaagccctg tgtaaatgga ttgattttac gcgcgatttt
     3361 ctcagcaaat acaccaagac cacttcaata gatttgagtt cgctgcgtcc gagctcccag
     3421 tataaagacc tgggcgaata ttatgccgag ctgaacccgt tactgtacca cattagcttc
     3481 cagcgcattg cggaaaaaga aatcatggat gcagtcgaaa ctggaaagct ttatctgttc
     3541 cagatttaca acaaggattt tgcgaaaggc catcacggca aaccgaatct acatactctc
     3601 tattggactg gcctgttctc tcccgaaaac ttggccaaga cctcaattaa actcaatggc
     3661 caagccgagc tgttctaccg tccgaaatcg cgcatgaagc ggatggccca tcgtcttggg
     3721 gagaaaatgc tcaataagaa actgaaggat cagaaaacac cgatccctga cacgctgtat
     3781 caggaacttt acgactatgt caatcaccgt ctgagtcacg atctgtcgga cgaagcacgg
     3841 gccttactgc cgaacgtaat aaccaaagag gtgtcacacg agatcattaa ggatcgacgt
     3901 tttacctccg acaaattctt tttccacgta ccgattaccc ttaattacca ggcggcgaac
     3961 tcaccctcga aatttaacca gcgcgtaaac gcgtacctga aagagcaccc ggaaacccct
     4021 attattggga ttgaccgtgg cgaacgcaat cttatctata taaccgtaat cgactctacc
     4081 ggcaagattt tagaacagcg tagcctaaac accattcagc aatttgatta ccagaaaaag
     4141 ttagataatc gggaaaaaga acgtgtggcg gcacggcagg cttggtcggt agttgggacg
     4201 attaaggatc ttaaacaagg ttatttatcc caggttatac acgaaatcgt agacttaatg
     4261 atacattatc aagccgttgt ggtgctggcc aacctgaact tcggatttaa gtcaaagcgg
     4321 acgggcattg ccgaaaaggc ggtgtatcag cagttcgaga agatgctgat cgataaactc
     4381 aattgcttag tactaaaaga ttatcccgct gaaaaggtag gtggcgttct caacccgtac
     4441 cagcttacag atcaatttac cagctttgcg aagatgggta cacagagcgg ttttctgttt
     4501 tatgttcccg caccgtatac cagcaaaatc gatcccctga ccggctttgt tgatcctttt
     4561 gtatggaaaa ccatcaaaaa ccacgaaagc cgcaaacatt tcctggaggg ctttgatttt
     4621 cttcactacg atgtgaaaac aggcgatttt attctgcact tcaaaatgaa tcgcaatctt
     4681 agctttcagc gcggtctgcc tgggtttatg ccggcgtggg atattgtgtt cgaaaagaat
     4741 gaaacgcagt tcgacgccaa aggcacaccg tttattgccg ggaaacgtat agtaccggtg
     4801 atcgaaaatc atcggtttac aggccgttat cgcgacctgt acccggctaa tgagctgatt
     4861 gcactgctcg aagaaaaggg aattgtattt cgtgacggtt ccaatattct gcctaaactg
     4921 ttggaaaatg atgactcgca cgcgattgat accatggtgg ctcttatccg cagcgttctt
     4981 caaatgcgta actctaacgc ggcgacgggg gaagattata ttaacagccc ggtacgcgat
     5041 ctcaacggcg tctgttttga tagtcgcttc cagaaccccg aatggccgat ggacgcggac
     5101 gccaacggag cataccatat tgcgctgaaa ggccagctgt tactgaatca tctcaaagaa
     5161 tccaaagacc tcaaactaca gaatggcatt tcaaatcagg attggctcgc gtatattcag
     5221 gaactgcgca attgatagtc catgtgacct tcaccctcat ggaccgcaaa aaaccccgct
     5281 tcggcggggt tttttcgctg cgcaactttg gtaacttgat gtctacacct aaatgagctg
     5341 ttgacaatta atcatccggc tcgtataatg tgtggaattg tgagcggata acaatttcaa
     5401 tttctactct tgtagataaa gttcgtatgg aaggttccgt tttttttgaa gcttgggccc
     5461 gaacaaaaac tcggatccaa actcgagtaa ggatctccag gcatcaaata aaacgaaagg
     5521 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact
     5581 agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatacctag ggtacgggtt
     5641 ttgctgcccg caaacgggct gttctggtgt tgctagtttg ttatcagaat cgcagatccg
     5701 gcttcagccg gtttgccggc tgaaagcgct atttcttcca gaattgccat gattttttcc
     5761 ccacgggagg cgtcactggc tcccgtgttg tcggcagctt tgattcgata agcagcatcg
     5821 cctgtttcag gctgtctatg tgtgactgtt gagctgtaac aagttgtctc aggtgttcaa
     5881 tttcatgttc tagttgcttt gttttactgg tttcacctgt tctattaggt gttacatgct
     5941 gttcatctgt tacattgtcg atctgttcat ggtgaacagc tttgaatgca ccaaaaactc
     6001 gtaaaagctc tgatgtatct atctttttta caccgttttc atctgtgcat atggacagtt
     6061 ttccctttga tatgtaacgg tgaacagttg ttctactttt gtttgttagt cttgatgctt
     6121 cactgataga tacaagagcc ataagaacct cagatccttc cgtatttagc cagtatgttc
     6181 tctagtgtgg ttcgttgttt ttgcgtgagc catgagaacg aaccattgag atcatactta
     6241 ctttgcatgt cactcaaaaa ttttgcctca aaactggtga gctgaatttt tgcagttaaa
     6301 gcatcgtgta gtgtttttct tagtccgtta tgtaggtagg aatctgatgt aatggttgtt
     6361 ggtattttgt caccattcat ttttatctgg ttgttctcaa gttcggttac gagatccatt
     6421 tgtctatcta gttcaacttg gaaaatcaac gtatcagtcg ggcggcctcg cttatcaacc
     6481 accaatttca tattgctgta agtgtttaaa tctttactta ttggtttcaa aacccattgg
     6541 ttaagccttt taaactcatg gtagttattt tcaagcatta acatgaactt aaattcatca
     6601 aggctaatct ctatatttgc cttgtgagtt ttcttttgtg ttagttcttt taataaccac
     6661 tcataaatcc tcatagagta tttgttttca aaagacttaa catgttccag attatatttt
     6721 atgaattttt ttaactggaa aagataaggc aatatctctt cactaaaaac taattctaat
     6781 ttttcgcttg agaacttggc atagtttgtc cactggaaaa tctcaaagcc tttaaccaaa
     6841 ggattcctga tttccacagt tctcgtcatc agctctctgg ttgctttagc taatacacca
     6901 taagcatttt ccctactgat gttcatcatc tgagcgtatt ggttataagt gaacgatacc
     6961 gtccgttctt tccttgtagg gttttcaatc gtggggttga gtagtgccac acagcataaa
     7021 attagcttgg tttcatgctc cgttaagtca tagcgactaa tcgctagttc atttgctttg
     7081 aaaacaacta attcagacat acatctcaat tggtctaggt gattttaatc actataccaa
     7141 ttgagatggg ctagtcaatg ataattacta gtccttttcc cgggtgatct gggtatctgt
     7201 aaattctgct agacctttgc tggaaaactt gtaaattctg ctagaccctc tgtaaattcc
     7261 gctagacctt tgtgtgtttt ttttgtttat attcaagtgg ttataattta tagaataaag
     7321 aaagaataaa aaaagataaa aagaatagat cccagccctg tgtataactc actactttag
     7381 tcagttccgc agtattacaa aaggatgtcg caaacgctgt ttgctcctct acaaaacaga
     7441 ccttaaaacc ctaaaggctt aagtagcacc ctcgcaagct cgggcaaatc gctgaatatt
     7501 ccttttgtct ccgaccatca ggcacctgag tcgctgtctt tttcgtgaca ttcagttcgc
     7561 tgcgctcacg gctctggcag tgaatggggg taaatggcac tacaggcgcc ttttatggat
     7621 tcatgcaagg aaactaccca taatacaaga aaagcccgtc acgggcttct cagggcgttt
     7681 tatggcgggt ctgctatgtg gtgctatctg actttttgct gttcagcagt tcctgccctc
     7741 tgattttcca gtctgaccac ttcggattat cccgtgacag gtcattcaga ctggctaatg
     7801 cacccagtaa ggcagcggta tcatcaacag gcttacccgt cttactgtcc ctagtgcttg
     7861 gattctcacc aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa tccagatgga
     7921 gttctgaggt cattactgga tctatcaaca ggagtccaag cgagctcgat atcaaattac
     7981 gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg
     8041 aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct
     8101 tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg
     8161 tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca
     8221 ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat
     8281 atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca
     8341 gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg
     8401 tctttcattg ccatacgaaa ttccggatga gcattcatca ggcgggcaag aatgtgaata
     8461 aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc
     8521 agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct
     8581 ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta
     8641 gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt
     8701 tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccagata
     8761 tc
//
