LOCUS       sequence-177282-        5594 bp    DNA     linear   UNA 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      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5594)
  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 5594)
  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..5594
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             48..1130
                     /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(67..86)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      1216..1804
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1705..1724
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1958..1975
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     2073..2090
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2074..2090
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2717..3430
                     /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             3449..3466
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3674..3693)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(3699..3718)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        3760..3851
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3852..3920,3921..4712)
                     /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(4070..4089)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             4758..5594
                     /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"
ORIGIN
        1 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatatg aaaccagtaa
       61 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
      121 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
      181 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
      241 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
      301 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
      361 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
      421 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
      481 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgagg
      541 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
      601 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
      661 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
      721 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
      781 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
      841 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga tagctcatgt tatatcccgc
      901 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
      961 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgccagtc tcactggtga
     1021 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     1081 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga ctcatgacca
     1141 aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc ccgtagaaaa
     1201 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1261 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1321 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     1381 gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1441 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1501 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1561 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1621 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1681 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1741 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1801 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     1861 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     1921 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     1981 ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc tgacggatgg
     2041 cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt cttaagctcg
     2101 ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg taaaaacccg
     2161 cttcggcggg tttttttatg gggggagttt agggaaagag catttgtcag aatatttaag
     2221 ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg agaaaaaagc
     2281 aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata attaaactat
     2341 tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa agagaattaa
     2401 gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa attatacatg
     2461 tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt attatgcatt
     2521 tagaataaat tttgtgtcgc ccttccacaa ttcagcaaat tgtgaacatc atcacgttca
     2581 tctttccctg gttgccaatg gcccattttc ctgtcagtaa cgagaaggtc gcgtattcag
     2641 gcgcttttta gactggtcgt aatgaaacgg tttccctcta gaaataattt tgtttaactt
     2701 ttaggaggta aaacatatgc gtaaaggcga agagctgttc actggtgtcg tccctattct
     2761 ggtggaactg gatggtgatg tcaacggtca taagttttcc gtgcgtggcg agggtgaagg
     2821 tgacgcaact aatggtaaac tgacgctgaa gttcatctgt actactggta aactgccggt
     2881 accttggccg actctggtaa cgacgctgac ttatggtgtt cagtgctttg ctcgttatcc
     2941 ggaccatatg aagcagcatg acttcttcaa gtccgccatg ccggaaggct atgtgcagga
     3001 acgcacgatt tcctttaagg atgacggcac gtacaaaacg cgtgcggaag tgaaatttga
     3061 aggcgatacc ctggtaaacc gcattgagct gaaaggcatt gactttaaag aagacggcaa
     3121 tatcctgggc cataagctgg aatacaattt taacagccac aatgtttaca tcaccgccga
     3181 taaacaaaaa aatggcatta aagcgaattt taaaattcgc cacaacgtgg aggatggcag
     3241 cgtgcagctg gctgatcact accagcaaaa cactccaatc ggtgatggtc ctgttctgct
     3301 gccagacaat cactatctga gcacgcaaag cgttctgtct aaagatccga acgagaaacg
     3361 cgatcatatg gttctgctgg agttcgtaac cgcagcgggc atcacgcatg gtatggatga
     3421 actgtacaaa ggctccggct ccggctccca ccatcaccat caccattgat aactcgagcc
     3481 ccaagggcga caccccataa ttagcccggg cgaaaggccc agtctttcga ctgagccttt
     3541 cgttttattt gatgcctggc agttccctac tctcgcatgg ggagtcccca cactaccatc
     3601 ggcgctacgg cgtttcactt ctgagttcgg catggggtca ggtgggacca ccgcgctact
     3661 gccgccaggc aaacaagggg tgttatgagc catattcagg tataaatggg ctcgcgataa
     3721 tgttcagaat tggttaattg gttgtaacac tgacccctat ttgtttattt ttctaaatac
     3781 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3841 aaaggaagaa tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3901 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3961 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4021 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4081 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4141 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4201 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4261 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4321 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4381 acaccacgat gcctgtagcg atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4441 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     4501 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatcc ggagccggtg
     4561 agcgtggttc tcgcggtatc atcgcagcgc tggggccaga tggtaagccc tcccgtatcg
     4621 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     4681 agataggtgc ctcactgatt aagcattggt aagcggcgcg ccatatttcg ccgtgttgac
     4741 gacatcagga ggccagtatg accgtattac atagtgtgga tttttttccg tctggtaacg
     4801 cgtccgtggc gatagaaccc cggctcccgc aggcggattt tcctgaacat catcatgatt
     4861 ttcatgaaat tgtgattgtc gaacatggca cgggtattca tgtgtttaat gggcagccct
     4921 ataccatcac cggtggcacg gtctgtttcg tacgcgatca tgatcggcat ctgtatgaac
     4981 ataccgataa tctgtgtctg accaatgtgc tgtatcgctc gccggatcga tttcagtttc
     5041 tcgccgggct gaatcagttg ctgccacaag agctggatgg gcagtatccg tctcactggc
     5101 gcgttaacca cagcgtattg cagcaggtgc gacagctggt tgcacagatg gaacagcagg
     5161 aaggggaaaa tgatttaccc tcgaccgcca gtcgcgagat cttgtttatg caattactgc
     5221 tcttgctgcg taaaagcagt ttgcaggaga acctggaaaa cagcgcatca cgtctcaact
     5281 tgcttctggc ctggctggag gaccattttg ccgatgaggt gaattgggat gccgtggcgg
     5341 atcaattttc tctttcactg cgtacgctac atcggcagct taagcagcaa acgggactga
     5401 cgcctcagcg atacctgaac cgcctgcgac tgatgaaagc ccgacatctg ctacgccaca
     5461 gcgaggccag cgttactgac atcgcctatc gctgtggatt cagcgacagt aaccactttt
     5521 cgacgctttt tcgccgagag tttaactggt caccgcgtga tattcgccag ggacgggatg
     5581 gctttctgca ataa
//
