LOCUS Exported 8762 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION For CRISPRi. Arabinose-inducible ddcpf1 gene with CRISPR array targeting the mrfp reporter gene.. ACCESSION . VERSION . KEYWORDS pBbS8c-ddcpf1-rfp 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8762 /organism="synthetic DNA construct" /mol_type="other DNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter 5341..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 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 //