LOCUS       sequence-171131-        5618 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  As pCK301 (E. coli rhaBAD promoter upstream of sfGFP, sfGFP can be 
            replaced with any gene of interest), but rhaS cloned downstream of 
            ampR, which allows non-metabolisable inducer L-mannose to be used.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5618)
  AUTHORS   Kelly CL, Liu Z, Yoshihara A, Jenkinson SF, Wormald MR, Otero J, 
            Estevez A, Kato A, Marqvorsen MH, Fleet GW, Estevez RJ, Izumori K, 
            Heap JT
  TITLE     Synthetic Chemical Inducers and Genetic Decoupling Enable Orthogonal
            Control of the rhaBAD Promoter.
  JOURNAL   ACS Synth Biol. 2016 Oct 21;5(10):1136-1145. Epub 2016 Jun 21.
  PUBMED    27247275
REFERENCE   2  (bases 1 to 5618)
  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: "ACS Synth 
            Biol."; date: "2016-10-21"; volume: "5(10)"; pages: "1136-1145. Epub
            2016 Jun 21"
COMMENT     Sequence Label: pCK302
FEATURES             Location/Qualifiers
     source          1..5618
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      86..674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     575..594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     828..845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     943..960
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     944..960
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1587..2300
                     /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             2319..2336
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2544..2563)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2569..2588)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2630..2721
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2722..2790,2791..3582)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2940..2959)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             3628..4464
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     CDS             4536..5618
                     /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     complement(4555..4574)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc
       61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
      841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc
      901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt
      961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg
     1021 taaaaacccg cttcggcggg tttttttatg gggggagttt agggaaagag catttgtcag
     1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg
     1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata
     1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa
     1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa
     1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt
     1381 attatgcatt tagaataaat tttgtgtcgc ccttccacaa ttcagcaaat tgtgaacatc
     1441 atcacgttca tctttccctg gttgccaatg gcccattttc ctgtcagtaa cgagaaggtc
     1501 gcgtattcag gcgcttttta gactggtcgt aatgaaacgg tttccctcta gaaataattt
     1561 tgtttaactt ttaggaggta aaacatatgc gtaaaggcga agagctgttc actggtgtcg
     1621 tccctattct ggtggaactg gatggtgatg tcaacggtca taagttttcc gtgcgtggcg
     1681 agggtgaagg tgacgcaact aatggtaaac tgacgctgaa gttcatctgt actactggta
     1741 aactgccggt accttggccg actctggtaa cgacgctgac ttatggtgtt cagtgctttg
     1801 ctcgttatcc ggaccatatg aagcagcatg acttcttcaa gtccgccatg ccggaaggct
     1861 atgtgcagga acgcacgatt tcctttaagg atgacggcac gtacaaaacg cgtgcggaag
     1921 tgaaatttga aggcgatacc ctggtaaacc gcattgagct gaaaggcatt gactttaaag
     1981 aagacggcaa tatcctgggc cataagctgg aatacaattt taacagccac aatgtttaca
     2041 tcaccgccga taaacaaaaa aatggcatta aagcgaattt taaaattcgc cacaacgtgg
     2101 aggatggcag cgtgcagctg gctgatcact accagcaaaa cactccaatc ggtgatggtc
     2161 ctgttctgct gccagacaat cactatctga gcacgcaaag cgttctgtct aaagatccga
     2221 acgagaaacg cgatcatatg gttctgctgg agttcgtaac cgcagcgggc atcacgcatg
     2281 gtatggatga actgtacaaa ggctccggct ccggctccca ccatcaccat caccattgat
     2341 aactcgagcc ccaagggcga caccccataa ttagcccggg cgaaaggccc agtctttcga
     2401 ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcatgg ggagtcccca
     2461 cactaccatc ggcgctacgg cgtttcactt ctgagttcgg catggggtca ggtgggacca
     2521 ccgcgctact gccgccaggc aaacaagggg tgttatgagc catattcagg tataaatggg
     2581 ctcgcgataa tgttcagaat tggttaattg gttgtaacac tgacccctat ttgtttattt
     2641 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2701 taatattgaa aaaggaagaa tatgagtatt caacatttcc gtgtcgccct tattcccttt
     2761 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     2821 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     2881 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     2941 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     3001 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     3061 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     3121 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3181 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     3241 gacgagcgtg acaccacgat gcctgtagcg atggcaacaa cgttgcgcaa actattaact
     3301 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3361 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatcc
     3421 ggagccggtg agcgtggttc tcgcggtatc atcgcagcgc tggggccaga tggtaagccc
     3481 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3541 cagatcgctg agataggtgc ctcactgatt aagcattggt aagcggcgcg ccatatttcg
     3601 ccgtgttgac gacatcagga ggccagtatg accgtattac atagtgtgga tttttttccg
     3661 tctggtaacg cgtccgtggc gatagaaccc cggctcccgc aggcggattt tcctgaacat
     3721 catcatgatt ttcatgaaat tgtgattgtc gaacatggca cgggtattca tgtgtttaat
     3781 gggcagccct ataccatcac cggtggcacg gtctgtttcg tacgcgatca tgatcggcat
     3841 ctgtatgaac ataccgataa tctgtgtctg accaatgtgc tgtatcgctc gccggatcga
     3901 tttcagtttc tcgccgggct gaatcagttg ctgccacaag agctggatgg gcagtatccg
     3961 tctcactggc gcgttaacca cagcgtattg cagcaggtgc gacagctggt tgcacagatg
     4021 gaacagcagg aaggggaaaa tgatttaccc tcgaccgcca gtcgcgagat cttgtttatg
     4081 caattactgc tcttgctgcg taaaagcagt ttgcaggaga acctggaaaa cagcgcatca
     4141 cgtctcaact tgcttctggc ctggctggag gaccattttg ccgatgaggt gaattgggat
     4201 gccgtggcgg atcaattttc tctttcactg cgtacgctac atcggcagct taagcagcaa
     4261 acgggactga cgcctcagcg atacctgaac cgcctgcgac tgatgaaagc ccgacatctg
     4321 ctacgccaca gcgaggccag cgttactgac atcgcctatc gctgtggatt cagcgacagt
     4381 aaccactttt cgacgctttt tcgccgagag tttaactggt caccgcgtga tattcgccag
     4441 ggacgggatg gctttctgca ataatcgaat ggcgcaaaac ctttcgcggt atggcatgat
     4501 agcgcccgga agagagtcaa ttcagggtgg tgaatatgaa accagtaacg ttatacgatg
     4561 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     4621 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     4681 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     4741 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     4801 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     4861 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     4921 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     4981 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaggac ggtacgcgac
     5041 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     5101 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     5161 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     5221 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     5281 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct
     5341 cggtagtggg atacgacgat accgaagata gctcatgtta tatcccgccg ttaaccacca
     5401 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     5461 agggccaggc ggtgaagggc aatcagctgt tgccagtctc actggtgaaa agaaaaacca
     5521 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     5581 tggcacgaca ggtttcccga ctggaaagcg ggcagtga
//
