LOCUS       sequence-177279-        4747 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli rhaBAD promoter upstream of sfGFP, sfGFP can be replaced 
            with any gene of interest to express using L-rhamnose, three 
            terminators, ampR, pBR322 origin, lacI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4747)
  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 4747)
  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: pCK301
FEATURES             Location/Qualifiers
     source          1..4747
                     /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             3665..4747
                     /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(3684..3703)
                     /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 ccatcgaatg
     3601 gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     3661 gaatatgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     3721 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     3781 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     3841 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     3901 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     3961 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     4021 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     4081 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     4141 tattttctcc catgaggacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     4201 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     4261 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     4321 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     4381 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     4441 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagatag
     4501 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     4561 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     4621 gccagtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     4681 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     4741 gcagtga
//
