LOCUS       sequence-484518-        7952 bp    DNA     circular SYN 25-AUG-2025
DEFINITION  Landing Pad with RhaRS/PrhaBAD, pC194+pKan+ptetQ seed promoter, 
            pcepA-himar transposase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7952)
  AUTHORS   Nakamura AK, Fulk EM, Johnson CW, Isaacs FJ
  TITLE     Synthetic Genetic Elements Enable Rapid Characterization of 
            Inorganic Carbon Uptake Systems in Cupriavidus necator H16.
  JOURNAL   ACS Synth Biol. 2025 Mar 21;14(3):943-953. doi: 
            10.1021/acssynbio.4c00869. Epub 2025 Mar 6.
  PUBMED    40048245
REFERENCE   2  (bases 1 to 7952)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acssynbio.4c00869"; journalName: "ACS Synth Biol"; date: 
            "2025-03-21- 21"; volume: "14"; issue: "3"; pages: "943-953"
COMMENT     Sequence Label: pLP-PrhaBAD-pcepA-himar-chlorR
FEATURES             Location/Qualifiers
     source          1..7952
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(47..165)
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             311..1147
                     /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             1221..2069
                     /codon_start=1
                     /gene="E. coli rhaR"
                     /product="transcriptional activator of rhaR and rhaS"
                     /label=rhaR
                     /note="needed for rhamnose-dependent activation of the rhaB
                     promoter"
                     /translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     promoter        2074..2108
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     CDS             2145..2804
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2211..2230)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2874..3262
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     oriT            5273..5381
                     /note="incP origin of transfer"
     terminator      complement(5528..5567)
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     promoter        complement(6432..6523)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        6580..6598
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6580..6597
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(join(7216..7926,7927..7929))
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
ORIGIN
        1 aagccccaac tgagagaact cacaggttac cccagttggg gatggatacg accagtctaa
       61 aaagcgcctg aattcgcgac cttctcgtta ctgacaggaa aatgggccat tggcaaccag
      121 ggaaagatga acgtgatgat gttcacaatt tgctgaattg tggtgatgtg atgctcaccg
      181 catttcctga aaattcacgc tgtatcttga aaaatcgacg ttttttacgt ggttttccgt
      241 cgaaaattta aggtaagaac ctgacctcgt gattactatt tcgccgtgtt gacgacatca
      301 ggaggccagt atgaccgtat tacatagtgt ggattttttt ccgtctggta acgcgtccgt
      361 ggcgatagaa ccccggctcc cgcaggcgga ttttcctgaa catcatcatg attttcatga
      421 aattgtgatt gtcgaacatg gcacgggtat tcatgtgttt aatgggcagc cctataccat
      481 caccggtggc acggtctgtt tcgtacgcga tcatgatcgg catctgtatg aacataccga
      541 taatctgtgt ctgaccaatg tgctgtatcg ctcgccggat cgatttcagt ttctcgccgg
      601 gctgaatcag ttgctgccac aagagctgga tgggcagtat ccgtctcact ggcgcgttaa
      661 ccacagcgta ttgcagcagg tgcgacagct ggttgcacag atggaacagc aggaagggga
      721 aaatgattta ccctcgaccg ccagtcgcga gatcttgttt atgcaattac tgctcttgct
      781 gcgtaaaagc agtttgcagg agaacctgga aaacagcgca tcacgtctca acttgcttct
      841 ggcctggctg gaggaccatt ttgccgatga ggtgaattgg gatgccgtgg cggatcaatt
      901 ttctctttca ctgcgtacgc tacatcggca gcttaagcag caaacgggac tgacgcctca
      961 gcgatacctg aaccgcctgc gactgatgaa agcccgacat ctgctacgcc acagcgaggc
     1021 cagcgttact gacatcgcct atcgctgtgg attcagcgac agtaaccact tttcgacgct
     1081 ttttcgccga gagtttaact ggtcaccgcg tgatattcgc cagggacggg atggctttct
     1141 gcaataacgc gaatcttctc aacgtatttg tacgccatat tgcgaataat caacttcgtt
     1201 ctctggccga ggtagccacg gtggcgcatc agttaaaact tctcaaagat gatttttttg
     1261 ccagcgacca gcaggcagtc gctgtggctg accgttatcc gcaagatgtc tttgctgaac
     1321 atacacatga tttttgtgag ctggtgattg tctggcgcgg taatggcctg catgtactca
     1381 acgatcgccc ttatcgcatt acccgtggcg atctctttta cattcatgct gacgataaac
     1441 actcctacgc ttccgttaac gatctggttt tgcagaatat tatttattgc ccggagcgtc
     1501 tgaagctgaa tcttgactgg cagggggcga ttccgggatt taacgccagc gcagggcaac
     1561 cacactggcg cttaggtagc atggggatgg cgcaggcgcg gcaggttatc ggtcagcttg
     1621 agcatgaaag tagtcagcat gtgccgtttg ctaacgaaat ggctgagttg ctgttcgggc
     1681 agttggtgat gttgctgaat cgccatcgtt acaccagtga ttcgttgccg ccaacatcca
     1741 gcgaaacgtt gctggataag ctgattaccc ggctggcggc tagcctgaaa agtccctttg
     1801 cgctggataa attttgtgat gaggcatcgt gcagtgagcg cgttttgcgt cagcaatttc
     1861 gccagcagac tggaatgacc atcaatcaat atctgcgaca ggtcagagtg tgtcatgcgc
     1921 aatatcttct ccagcatagc cgcctgttaa tcagtgatat ttcgaccgaa tgtggctttg
     1981 aagatagtaa ctatttttcg gtggtgttta cccgggaaac cgggatgacg cccagccagt
     2041 ggcgtcatct caattcgcag aaagattaaa tctttgacag ctagctcagt cctaggtata
     2101 atgctagcac ggaaaaaagg atctattata aggaggcact caccatggag aaaaaaatca
     2161 ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc
     2221 agtcagttgc tcaatgtacc tataaccaga ccgttcaact ggatattacg gcctttttaa
     2281 agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc
     2341 tgatgaatgc tcatccggaa ttacgtatgg caatgaaaga cggtgagctg gtgatatggg
     2401 atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct
     2461 ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt
     2521 gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct
     2581 cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact
     2641 tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc
     2701 cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta
     2761 atgaattaca acagtactgc gatgagtggc agggaggagc gtaactcgtt tgttgaacta
     2821 atgggtgctc agaccgggga cttatcatcc aacctgttac tgggcgcgcc ccatgtcagc
     2881 cgttaagtgt tcctgtgtca ctcaaaattg ctttgagagg ctctaagggc ttctcagtgc
     2941 gttacatccc tggcttgttg tccacaaccg ttaaacctta aaggctttaa aagccttata
     3001 tattcttttt tttcttataa aacttaaaac cttagaggct atttaagttg ctgatttata
     3061 ttaattttat tgttcaaaca tgagagctta gtacgtgaaa catgagagct tagtacgtta
     3121 gccatgagag cttagtacgt tagccatgag ggtttagttc gttaaacatg agagcttagt
     3181 acgttaaaca tgagagctta gtacgtgaaa catgagagct tagtacgtac tatcaacagg
     3241 ttgaactgct gatcttcaga tcggcgcgcc ggccggcatc ttgccgtaac tgacaaatta
     3301 accgaaggtt tcttccggcc actgggatgc aatgacctta ccaacaacgc tacaggattc
     3361 gttgcacggg atcattgggt attgcggatt cagcggctgc aggaaaactt ggccagagtc
     3421 gcggatcagc ttcttaaagg tgaattcatc accacccagg cgcgcaatgc aaaagtctcc
     3481 cggctcaacc gcctgctcag gatcaaccag gatcagcata ccatccggaa atgacggctt
     3541 gctgcccgtc ggggccgtca tgctgttgcc ttctacttcc agccaaaacg cgctatcgct
     3601 cgcttttttg gtagtcgata cccaacgctc agcgtcacct ttggtgaagg tacgcagttc
     3661 cgggctaaac atacctgctt gaacgtggct gaagaccggg tattcgtatt cagagcgcag
     3721 agacggttgc atgctaaccg cttcatacat ttcatagatt tcacgagcta tagaaggaga
     3781 gaactcctcg acggaaactt tcagaatctt ggtcagcagt gcggcgttgt acgcgttcag
     3841 cgcattaata ccgttaaaca gagcgccaac gccgctctga cccatgccca tcttgtcggc
     3901 tacgctttcc tgagacagac ccagctcatt ctttttcttc tcatagattg ctttcagacg
     3961 acgtgcgtcc tctagctgct cttgtgtaag cggctttttc ttcgttgaca ttttgatcct
     4021 cctttatatg gaggtactaa atcggaacgt taaatctatc accgcaaggg ataaatatct
     4081 aacaccgtgc gtgttgacta ttttacctct ggcggtgata atggttgcat caaatttgcg
     4141 cgccacatta ttattcatac ctttgtggac cgtattacaa agtgacaata tcttaattta
     4201 aaaaggaggc ttaaataatg gaaaaaaaag aatttagggt acttataaaa tactgctttc
     4261 tgaagggtaa gaacaccgtc gaagcaaaga cgtggctgga taacgaattt ccggacagcg
     4321 ccccgggtaa atcaaccatt atcgactggt acgccaagtt taagcgtggt gaaatgtcga
     4381 ccgaagatgg tgagcgttcc ggtcgtccga aggaggttgt caccgacgaa aatataaaaa
     4441 aaattcacaa aatgattctg aacgaccgca aaatgaagct gatcgaaatt gcggaagctc
     4501 tgaaaattag caaagagcgc gttggtcaca tcatccacca atatcttgac atgcgtaaac
     4561 tgtgtgcgaa atgggttccg cgcgaactga cctttgatca gaaacagcgt cgtgtcgacg
     4621 attctaagcg ttgcctgcag ctgctgaccc gcaacacgcc ggagttcttc cgccgctacg
     4681 taaccatgga tgagacgtgg ctgcaccact ataccccgga gtctaaccgt cagtcagcag
     4741 aatggacggc aactggcgaa ccgagcccga aacgcggcaa aacccaaaag agcgcgggca
     4801 aagtcatggc gagcgtattt tgggatgcac atggtattat cttcattgac tacctggaaa
     4861 agggtaagac cataaattcc gattactata tggcgctgct ggaacgcctg aaagttgaaa
     4921 tcgcggcaaa acgcccgcac atgaaaaaga agaaggtact gtttcaccag gacaacgccc
     4981 cctgtcataa gtccctgcgt accatggcga agattcatga gctgggtttc gaactgctgc
     5041 cgcacccgcc gtacagcccg gatctggctc cgtcggattt ttttctgttt agcgacctga
     5101 agcgtatgct ggcgggtaaa aaatttggtt gcaacgaaga agttatcgct gaaaccgaag
     5161 cgtactttga agcgaagccg aaagaatatt accagaacgg cattaagaaa ctggaaggtc
     5221 gttacaatcg ctgtatcgcg ctggagggca attacgtaga gtaaccggcc tacggccagc
     5281 ctcacagagc aggattcccg ttgagcaccg ccaggtgcga ataagggaca gtgaagaagg
     5341 aacacccgct cgcgggtggg cctacttcac ctatcctgcc cggctgacgc cgttggatac
     5401 accaaggaaa gtctacacga accctttggc aaaatcctgt atatcgtgcg aaaaaggatg
     5461 gatataccga aaaaatcgct ataatgaccc cgaagcaggg ttatgcagcg gaaaaggaca
     5521 agggtcgtcc aaaaaaaaag gctccaaaag gagcctttaa ttgtatcggt ttaccaatgt
     5581 ttaatcaggc tcgcgccaat ttccgcaatc tgacggttac gttcatccat cgtcgcctgg
     5641 ctgccggtgg tataaatcac cacaatacgg ctcggtttac catccgggcc cagcgccgca
     5701 ataatgccac ggctgccacg ttcgcccgca ccgcttttat ccgcaataaa ccagccagcc
     5761 ggcagcgcgc tacgcagcag cggacccgcc actttatccg cttccatcca atcaatcagt
     5821 tgctggcgac ttgccagggt cagcagctca cccgtcagca gtttacgcag ggtggtcgcc
     5881 attgctgccg gcatggtggt atcacgttca tcgttcggaa tcgcttcgtt cagttccggt
     5941 tcccaacgat ccagacgggt cacatgatcg cccatgttat gcagaaacgc ggtcagttct
     6001 ttcggaccgc caatggtcgt cagcagcagg ttcgccgcgg tgttatcgct catggtaatc
     6061 gccgcgctgc acagttcacg cacggtcatg ccattggtca gatgtttttc ggtcaccggg
     6121 ctatattcca ccagatcgtt ctggctataa tgaatacgac ggcccagttg ttcctggccc
     6181 gcatccacac ggctcagaac cgcaccgcac agcagcactt taaaggtgct catcatcgga
     6241 aaacgttctt ccggacgaaa agattccaga attttgccgc tgttcagatc cagttcaata
     6301 tagcccacgc gcgcacccag ttgatcttcc gcatctttca ctttcaccag ggtttccgga
     6361 tgcgcaaaca ccggtaggca aaacgccgca aaaaaaggaa tcagcgccac acgaaaatgc
     6421 tgaatgctca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6481 atgagcggat acatatttga atgtatttag aaaaataaac aaatagggga attaaaaaaa
     6541 agcccgctca ttaggcgggc tactaattta aatcgattaa tttaggtgac actatagaca
     6601 gtaacaggtt ggatgataag tccccggtct actaccccaa ctgagagaac tctgaggtta
     6661 ccccagttgg ggtctagatt atgccaggat gcgctcgcac agacgccaac ccgtgacacc
     6721 gttgatggtc acgcggaaca gcaggctgcc gtccgggtta atcagacgct cgtcaataat
     6781 cttattgcca ttccacaggg taccggtgac ggtgatcttt ttaccgtcaa aaacggcaat
     6841 gccttcatac ggacggccga aatagtcaat catattcggg gtcacaccat caatcaccag
     6901 cgtaccgtaa tgcaagataa ccttgaagtg gtgatcgtca actgggtaga cgactttaaa
     6961 gattttctcg atttgaccca tttggtcacc gctcaggcct tcgtacggga tgatcacatg
     7021 aatatcaatt ttcagaccgt tctcgccgga cagaacgatg cgctgaatcg gcgtcacgct
     7081 cacacccagg ttctggaaca gagagcttac gccaccctgt tccaggactt gatccaaatt
     7141 ataacccgcg gtctgacgcc aatcgccaac aaaatcttcc agagtaaaaa ccataccgcc
     7201 cgatatatct gagcctttat acagttcgtc cataccgtgc gtgatgccgg ccgcggtcac
     7261 gaattccagc aggaccatgt ggtcacgctt ttcgttcgga tctttactca gcacagactg
     7321 cgtgctcagg taatgattgt ccggcagcag gaccgggcca tcaccaatcg gggtgttttg
     7381 ctgatagtgg tcggccagtt gaacgctgcc atcttccaca ttatggcgga tcttgaagtt
     7441 agccttgata ccgttcttct gcttatctgc ggtgatgtac acgttgtgcg aattgaagtt
     7501 atattccagt ttatggccca gaatattacc gtcttcctta aaatcgatgc ctttcagttc
     7561 aatacggtta accagcgtat caccttcgaa tttcacttcg gcgcgcgttt tgtaggtgcc
     7621 gtcatcttta aatgagatgg tacgttcttg gacataacct tccggcatcg cggacttgaa
     7681 aaagtcgtgg cgtttcatgt gatccgggta acgactgaaa cactgaacac catacgtcag
     7741 cgtggtgacc agggtcggcc acggaaccgg cagtttaccc gtggtgcaaa taaacttcag
     7801 cgtcagttta ccattggttg catcgccttc accttcgccg cgcacgctga atttgtgacc
     7861 attgacatca ccatccagtt ccaccagaat cggcaccacg cccgtaaaca gttcttcgcc
     7921 cttgctcata ttttttcctc cttatactta ag
//
