LOCUS       sequence-468911-        7952 bp    DNA     circular SYN 15-APR-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 Apr 15, 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(902..993)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1050..1068
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1050..1067
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(join(1686..2396,2397..2399))
                     /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"
     promoter        complement(2469..2587)
                     /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             2733..3569
                     /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             3643..4491
                     /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        4496..4530
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     CDS             4567..5226
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4633..4652)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      5296..5684
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     oriT            7695..7803
                     /note="incP origin of transfer"
     terminator      complement(join(7950..7952,1..37))
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
ORIGIN
        1 aaaaaaaaag gctccaaaag gagcctttaa ttgtatcggt ttaccaatgt ttaatcaggc
       61 tcgcgccaat ttccgcaatc tgacggttac gttcatccat cgtcgcctgg ctgccggtgg
      121 tataaatcac cacaatacgg ctcggtttac catccgggcc cagcgccgca ataatgccac
      181 ggctgccacg ttcgcccgca ccgcttttat ccgcaataaa ccagccagcc ggcagcgcgc
      241 tacgcagcag cggacccgcc actttatccg cttccatcca atcaatcagt tgctggcgac
      301 ttgccagggt cagcagctca cccgtcagca gtttacgcag ggtggtcgcc attgctgccg
      361 gcatggtggt atcacgttca tcgttcggaa tcgcttcgtt cagttccggt tcccaacgat
      421 ccagacgggt cacatgatcg cccatgttat gcagaaacgc ggtcagttct ttcggaccgc
      481 caatggtcgt cagcagcagg ttcgccgcgg tgttatcgct catggtaatc gccgcgctgc
      541 acagttcacg cacggtcatg ccattggtca gatgtttttc ggtcaccggg ctatattcca
      601 ccagatcgtt ctggctataa tgaatacgac ggcccagttg ttcctggccc gcatccacac
      661 ggctcagaac cgcaccgcac agcagcactt taaaggtgct catcatcgga aaacgttctt
      721 ccggacgaaa agattccaga attttgccgc tgttcagatc cagttcaata tagcccacgc
      781 gcgcacccag ttgatcttcc gcatctttca ctttcaccag ggtttccgga tgcgcaaaca
      841 ccggtaggca aaacgccgca aaaaaaggaa tcagcgccac acgaaaatgc tgaatgctca
      901 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
      961 acatatttga atgtatttag aaaaataaac aaatagggga attaaaaaaa agcccgctca
     1021 ttaggcgggc tactaattta aatcgattaa tttaggtgac actatagaca gtaacaggtt
     1081 ggatgataag tccccggtct actaccccaa ctgagagaac tctgaggtta ccccagttgg
     1141 ggtctagatt atgccaggat gcgctcgcac agacgccaac ccgtgacacc gttgatggtc
     1201 acgcggaaca gcaggctgcc gtccgggtta atcagacgct cgtcaataat cttattgcca
     1261 ttccacaggg taccggtgac ggtgatcttt ttaccgtcaa aaacggcaat gccttcatac
     1321 ggacggccga aatagtcaat catattcggg gtcacaccat caatcaccag cgtaccgtaa
     1381 tgcaagataa ccttgaagtg gtgatcgtca actgggtaga cgactttaaa gattttctcg
     1441 atttgaccca tttggtcacc gctcaggcct tcgtacggga tgatcacatg aatatcaatt
     1501 ttcagaccgt tctcgccgga cagaacgatg cgctgaatcg gcgtcacgct cacacccagg
     1561 ttctggaaca gagagcttac gccaccctgt tccaggactt gatccaaatt ataacccgcg
     1621 gtctgacgcc aatcgccaac aaaatcttcc agagtaaaaa ccataccgcc cgatatatct
     1681 gagcctttat acagttcgtc cataccgtgc gtgatgccgg ccgcggtcac gaattccagc
     1741 aggaccatgt ggtcacgctt ttcgttcgga tctttactca gcacagactg cgtgctcagg
     1801 taatgattgt ccggcagcag gaccgggcca tcaccaatcg gggtgttttg ctgatagtgg
     1861 tcggccagtt gaacgctgcc atcttccaca ttatggcgga tcttgaagtt agccttgata
     1921 ccgttcttct gcttatctgc ggtgatgtac acgttgtgcg aattgaagtt atattccagt
     1981 ttatggccca gaatattacc gtcttcctta aaatcgatgc ctttcagttc aatacggtta
     2041 accagcgtat caccttcgaa tttcacttcg gcgcgcgttt tgtaggtgcc gtcatcttta
     2101 aatgagatgg tacgttcttg gacataacct tccggcatcg cggacttgaa aaagtcgtgg
     2161 cgtttcatgt gatccgggta acgactgaaa cactgaacac catacgtcag cgtggtgacc
     2221 agggtcggcc acggaaccgg cagtttaccc gtggtgcaaa taaacttcag cgtcagttta
     2281 ccattggttg catcgccttc accttcgccg cgcacgctga atttgtgacc attgacatca
     2341 ccatccagtt ccaccagaat cggcaccacg cccgtaaaca gttcttcgcc cttgctcata
     2401 ttttttcctc cttatactta agaagcccca actgagagaa ctcacaggtt accccagttg
     2461 gggatggata cgaccagtct aaaaagcgcc tgaattcgcg accttctcgt tactgacagg
     2521 aaaatgggcc attggcaacc agggaaagat gaacgtgatg atgttcacaa tttgctgaat
     2581 tgtggtgatg tgatgctcac cgcatttcct gaaaattcac gctgtatctt gaaaaatcga
     2641 cgttttttac gtggttttcc gtcgaaaatt taaggtaaga acctgacctc gtgattacta
     2701 tttcgccgtg ttgacgacat caggaggcca gtatgaccgt attacatagt gtggattttt
     2761 ttccgtctgg taacgcgtcc gtggcgatag aaccccggct cccgcaggcg gattttcctg
     2821 aacatcatca tgattttcat gaaattgtga ttgtcgaaca tggcacgggt attcatgtgt
     2881 ttaatgggca gccctatacc atcaccggtg gcacggtctg tttcgtacgc gatcatgatc
     2941 ggcatctgta tgaacatacc gataatctgt gtctgaccaa tgtgctgtat cgctcgccgg
     3001 atcgatttca gtttctcgcc gggctgaatc agttgctgcc acaagagctg gatgggcagt
     3061 atccgtctca ctggcgcgtt aaccacagcg tattgcagca ggtgcgacag ctggttgcac
     3121 agatggaaca gcaggaaggg gaaaatgatt taccctcgac cgccagtcgc gagatcttgt
     3181 ttatgcaatt actgctcttg ctgcgtaaaa gcagtttgca ggagaacctg gaaaacagcg
     3241 catcacgtct caacttgctt ctggcctggc tggaggacca ttttgccgat gaggtgaatt
     3301 gggatgccgt ggcggatcaa ttttctcttt cactgcgtac gctacatcgg cagcttaagc
     3361 agcaaacggg actgacgcct cagcgatacc tgaaccgcct gcgactgatg aaagcccgac
     3421 atctgctacg ccacagcgag gccagcgtta ctgacatcgc ctatcgctgt ggattcagcg
     3481 acagtaacca cttttcgacg ctttttcgcc gagagtttaa ctggtcaccg cgtgatattc
     3541 gccagggacg ggatggcttt ctgcaataac gcgaatcttc tcaacgtatt tgtacgccat
     3601 attgcgaata atcaacttcg ttctctggcc gaggtagcca cggtggcgca tcagttaaaa
     3661 cttctcaaag atgatttttt tgccagcgac cagcaggcag tcgctgtggc tgaccgttat
     3721 ccgcaagatg tctttgctga acatacacat gatttttgtg agctggtgat tgtctggcgc
     3781 ggtaatggcc tgcatgtact caacgatcgc ccttatcgca ttacccgtgg cgatctcttt
     3841 tacattcatg ctgacgataa acactcctac gcttccgtta acgatctggt tttgcagaat
     3901 attatttatt gcccggagcg tctgaagctg aatcttgact ggcagggggc gattccggga
     3961 tttaacgcca gcgcagggca accacactgg cgcttaggta gcatggggat ggcgcaggcg
     4021 cggcaggtta tcggtcagct tgagcatgaa agtagtcagc atgtgccgtt tgctaacgaa
     4081 atggctgagt tgctgttcgg gcagttggtg atgttgctga atcgccatcg ttacaccagt
     4141 gattcgttgc cgccaacatc cagcgaaacg ttgctggata agctgattac ccggctggcg
     4201 gctagcctga aaagtccctt tgcgctggat aaattttgtg atgaggcatc gtgcagtgag
     4261 cgcgttttgc gtcagcaatt tcgccagcag actggaatga ccatcaatca atatctgcga
     4321 caggtcagag tgtgtcatgc gcaatatctt ctccagcata gccgcctgtt aatcagtgat
     4381 atttcgaccg aatgtggctt tgaagatagt aactattttt cggtggtgtt tacccgggaa
     4441 accgggatga cgcccagcca gtggcgtcat ctcaattcgc agaaagatta aatctttgac
     4501 agctagctca gtcctaggta taatgctagc acggaaaaaa ggatctatta taaggaggca
     4561 ctcaccatgg agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt
     4621 aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcaa
     4681 ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc
     4741 tttattcaca ttcttgcccg cctgatgaat gctcatccgg aattacgtat ggcaatgaaa
     4801 gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa
     4861 actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac
     4921 atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt
     4981 attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta
     5041 aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg
     5101 caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc
     5161 ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggagga
     5221 gcgtaactcg tttgttgaac taatgggtgc tcagaccggg gacttatcat ccaacctgtt
     5281 actgggcgcg ccccatgtca gccgttaagt gttcctgtgt cactcaaaat tgctttgaga
     5341 ggctctaagg gcttctcagt gcgttacatc cctggcttgt tgtccacaac cgttaaacct
     5401 taaaggcttt aaaagcctta tatattcttt tttttcttat aaaacttaaa accttagagg
     5461 ctatttaagt tgctgattta tattaatttt attgttcaaa catgagagct tagtacgtga
     5521 aacatgagag cttagtacgt tagccatgag agcttagtac gttagccatg agggtttagt
     5581 tcgttaaaca tgagagctta gtacgttaaa catgagagct tagtacgtga aacatgagag
     5641 cttagtacgt actatcaaca ggttgaactg ctgatcttca gatcggcgcg ccggccggca
     5701 tcttgccgta actgacaaat taaccgaagg tttcttccgg ccactgggat gcaatgacct
     5761 taccaacaac gctacaggat tcgttgcacg ggatcattgg gtattgcgga ttcagcggct
     5821 gcaggaaaac ttggccagag tcgcggatca gcttcttaaa ggtgaattca tcaccaccca
     5881 ggcgcgcaat gcaaaagtct cccggctcaa ccgcctgctc aggatcaacc aggatcagca
     5941 taccatccgg aaatgacggc ttgctgcccg tcggggccgt catgctgttg ccttctactt
     6001 ccagccaaaa cgcgctatcg ctcgcttttt tggtagtcga tacccaacgc tcagcgtcac
     6061 ctttggtgaa ggtacgcagt tccgggctaa acatacctgc ttgaacgtgg ctgaagaccg
     6121 ggtattcgta ttcagagcgc agagacggtt gcatgctaac cgcttcatac atttcataga
     6181 tttcacgagc tatagaagga gagaactcct cgacggaaac tttcagaatc ttggtcagca
     6241 gtgcggcgtt gtacgcgttc agcgcattaa taccgttaaa cagagcgcca acgccgctct
     6301 gacccatgcc catcttgtcg gctacgcttt cctgagacag acccagctca ttctttttct
     6361 tctcatagat tgctttcaga cgacgtgcgt cctctagctg ctcttgtgta agcggctttt
     6421 tcttcgttga cattttgatc ctcctttata tggaggtact aaatcggaac gttaaatcta
     6481 tcaccgcaag ggataaatat ctaacaccgt gcgtgttgac tattttacct ctggcggtga
     6541 taatggttgc atcaaatttg cgcgccacat tattattcat acctttgtgg accgtattac
     6601 aaagtgacaa tatcttaatt taaaaaggag gcttaaataa tggaaaaaaa agaatttagg
     6661 gtacttataa aatactgctt tctgaagggt aagaacaccg tcgaagcaaa gacgtggctg
     6721 gataacgaat ttccggacag cgccccgggt aaatcaacca ttatcgactg gtacgccaag
     6781 tttaagcgtg gtgaaatgtc gaccgaagat ggtgagcgtt ccggtcgtcc gaaggaggtt
     6841 gtcaccgacg aaaatataaa aaaaattcac aaaatgattc tgaacgaccg caaaatgaag
     6901 ctgatcgaaa ttgcggaagc tctgaaaatt agcaaagagc gcgttggtca catcatccac
     6961 caatatcttg acatgcgtaa actgtgtgcg aaatgggttc cgcgcgaact gacctttgat
     7021 cagaaacagc gtcgtgtcga cgattctaag cgttgcctgc agctgctgac ccgcaacacg
     7081 ccggagttct tccgccgcta cgtaaccatg gatgagacgt ggctgcacca ctataccccg
     7141 gagtctaacc gtcagtcagc agaatggacg gcaactggcg aaccgagccc gaaacgcggc
     7201 aaaacccaaa agagcgcggg caaagtcatg gcgagcgtat tttgggatgc acatggtatt
     7261 atcttcattg actacctgga aaagggtaag accataaatt ccgattacta tatggcgctg
     7321 ctggaacgcc tgaaagttga aatcgcggca aaacgcccgc acatgaaaaa gaagaaggta
     7381 ctgtttcacc aggacaacgc cccctgtcat aagtccctgc gtaccatggc gaagattcat
     7441 gagctgggtt tcgaactgct gccgcacccg ccgtacagcc cggatctggc tccgtcggat
     7501 ttttttctgt ttagcgacct gaagcgtatg ctggcgggta aaaaatttgg ttgcaacgaa
     7561 gaagttatcg ctgaaaccga agcgtacttt gaagcgaagc cgaaagaata ttaccagaac
     7621 ggcattaaga aactggaagg tcgttacaat cgctgtatcg cgctggaggg caattacgta
     7681 gagtaaccgg cctacggcca gcctcacaga gcaggattcc cgttgagcac cgccaggtgc
     7741 gaataaggga cagtgaagaa ggaacacccg ctcgcgggtg ggcctacttc acctatcctg
     7801 cccggctgac gccgttggat acaccaagga aagtctacac gaaccctttg gcaaaatcct
     7861 gtatatcgtg cgaaaaagga tggatatacc gaaaaaatcg ctataatgac cccgaagcag
     7921 ggttatgcag cggaaaagga caagggtcgt cc
//
