LOCUS       sequence-464926-        6441 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CCP expression in diverse gram-negative bacteria, with pre-cloned 
            Golden Gate Compatible (BsaI) restriction sites to facilitate 
            promoter swapping. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 2560 to 2594)
  AUTHORS   Armstrong ZR, Alonso J, Stanton V, Patel N, Zogaj X, Cocioba SS, 
            Klose KE
  TITLE     Mobilizable shuttle vectors with fluorescent markers functional 
            across different species of bacteria.
  JOURNAL   Appl Environ Microbiol. 2025 May 12:e0004525. doi: 
            10.1128/aem.00045-25.
  PUBMED    40353662
REFERENCE   2  (bases 1 to 6441)
  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: "Appl 
            Environ Microbiol. 2025 May 12:e0004525. doi: 10.1128/aem.00045-25."
COMMENT     Sequence Label: pKEK3232
FEATURES             Location/Qualifiers
     source          1..6441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1804..1821)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1938..2483)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2018..2037)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2537..2553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2560..2594
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     RBS             2607..2618
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(3367..3383)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3367..3383)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3575..4390
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             3575..4390
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3648..3667)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(5005..5376)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            5409..5518
                     /note="incP origin of transfer"
ORIGIN
        1 aatcctctgt aagtattgct acataaggct ttgaagcact tttttaaaaa atttaaaact
       61 actttttgta tcactttttg aatttaaaac tactttttgt atcacttttt gaatttaaaa
      121 ctactttttg tatcgttaaa actacttttt tttatgaagt agttgtttta ctaatagaag
      181 ttttatagaa tttgtatatc gagataatga ctctagttct atgaaagaag ttaaaaatgt
      241 aaatgctggc aaagaaatag ctatgtctaa tactcttgtt gcaggaaagt atagccttac
      301 aaaagaagaa caaaacttaa tattcttagt tgcctcaatg gttaaaagag aagataaaga
      361 gtttcacagg tataagataa gcctatctga tcttgaaaaa gctacagggg ttaagcataa
      421 cagagtgaga ttgaagcaat taatgcactc aataatgtct aagccagtat ggctgaataa
      481 agaacaaacg aagatagcta actggtttgc atatattgaa gcagacccta aaagcagtgc
      541 tttaatttgc gagtttcatt ggtcattaat gccacacatg atacaactac aagagtactt
      601 tactaaagca gaaagacagt tactattttc atttaaaagt aagtattcaa gcagattata
      661 cttacttctc aaaagcaaat taggcgaaca agcagggtat gctaatatag ttgattgcgt
      721 attgtatgtc gatgatatga ttaatgattt tgatttacct aaaagttaca gcaacagata
      781 ttcaaatttt aaaaataagt tcctattacc agcactagaa gagattaatc aattatcgga
      841 tatttttgta acctatgatg acaaggatca tcacagaaaa catggtcgta agattacaaa
      901 tatcaagttt acagttagta aggtagcaga gacagaagaa gaatttaagc aaaagccttt
      961 agatactaaa aataaagagg attacatacc aatcgaagca tctgaaagac ttagagaagt
     1021 tgtgttaagt gatgaactag acctagatct gtattatgtc agatcaatat ttagacatca
     1081 tcatttacat gatattgaaa gggtttgtga tgatacttgg aggaattggg acaataaaaa
     1141 aatcaaagat agaaaaagag tttttattgc aaatataaaa aaattagaaa agaaaccaga
     1201 agagaatcat aagctaccat ttggatttga ggaagtgtag gaatgaatat agaaatattt
     1261 aaattttcaa aaaaacataa aaaaataaat aagaagttaa tagatttaac aaaagtaaaa
     1321 caggttgtta ctactggcta tagctttatt gttgtatatc aaagaggtcg aaagatagag
     1381 ttaagttatg aaagttctta ctggcatgct aaaaaagatt tggataatag taatagtgac
     1441 gttggaataa catatagtta cattaaaaaa gataaaagag gcccctgtca caaagataac
     1501 tatttacgag actctaaagg tagactatta aaggactggg gaaaaataga agagcttcat
     1561 gaaaagtatc ctaaaatagg taaaatttgg tacatttcta gctttagctg tccctcctgt
     1621 tcagctactg acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg
     1681 agtgtatact ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg
     1741 caggagaaaa aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc
     1801 ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta
     1861 cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg
     1921 gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga
     1981 cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2041 ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg
     2101 ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc
     2161 caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa
     2221 ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg
     2281 taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga
     2341 caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc
     2401 agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta
     2461 cgcgcagacc aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga
     2521 gtagtcgact gagaccgtaa aacgacggcc agtgaattct tgacggctag ctcagtccta
     2581 ggtacagtgc tagcttggtc tcgattaaag aggagaaaga gtcccatggc gagcttagtc
     2641 aagaaagaca tgtgtgtaaa gatgacgatg gaggggactg ttaatggtta ccactttaag
     2701 tgtgtggggg aaggtgaggg gaaaccgttt gagggaactc aaaacatgcg tattcgcgtc
     2761 acggagggtg gacctttacc gttcgctttc gatattctgg caccatgttg catgtatggg
     2821 agcaaaacct ttattaaaca tgtcagcggc atcccagact attttaaaga gtcgttcccc
     2881 gaagggttca cctgggagcg tacacagatc ttcgaggatg gcggagtatt gaccgcacat
     2941 caggatacaa gtcttgaggg caattgcttg atttacaaag tgaaagtgct tgggacgaat
     3001 ttccctgcca acggacccgt tatgcaaaag aaaactgcag gctgggagcc gtgtgttgag
     3061 atgctttacc cacgtgacgg ggtattgtgc ggccaaagtc tgatggcact gaaatgtacc
     3121 gatggtaatc acttaacgtc gcacttacgt acaacatacc gtagccgtaa gccctccaac
     3181 gcggtcaaca tgcctgagtt tcactttggc gaccaccgca ttgagatctt aaaggcggaa
     3241 caggggaaat tttatgaaca gtatgagtcg gctgtggccc gttactctga tgtgcctgag
     3301 aaagcaactt aatgagagct caaaaaaaaa ccccgcccct gacagggcgg ggtttttttt
     3361 ggtaccgtca tagctgtttc ctgctcccca acggcgtaga ggatcgagat cttttgggtt
     3421 gtcactcatc gtatttggtt tataatttta agctaataac ctaattataa ctaattaata
     3481 gttttgtatc ttgaaaaaat agctataaaa cttatttaaa taacgaagat ttttgtgtat
     3541 aaaatattta taacaaaaaa aggagactaa acatatgagc catattcaac gggaaacgtc
     3601 ttgctcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     3661 tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc
     3721 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     3781 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     3841 tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt
     3901 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     3961 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     4021 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     4081 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataagcttt tgccattctc
     4141 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     4201 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     4261 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     4321 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     4381 gtttttctaa ggatccggct gctaacaaag cgcgagcagc cgaccaggct ttccacgccc
     4441 gcgtgccgct ccatgtcgtt cgcgcggttc tcggaaacgc gctgccgcgt ttcgtgattg
     4501 tcacgctcaa gcccgtagtc ccgttcgagc gtcgcgcaga ggtcagcgag ggcgcggtag
     4561 gcccgatacg gctcatggat ggtgtttcgg gtcgggtgaa tcttgttgat ggcgatatgg
     4621 atgtgcaggt tgtcggtgtc gtgatgcacg gcactgacgc gctgatgctc ggcgaagcca
     4681 agcccagcgc agatgcggtc ctcaatcgcg cgcaacgtct ccgcgtcggg cttctctccc
     4741 gcgcggaagc taaccaggag gtgataggtc ttgtcggcct cggaacgggt gttgccgtgc
     4801 tgggtcgcca tcacctcggc catgacagcg ggcagggtgt ttgcctcgca gttcgtgacg
     4861 cgcacgtgac ccaggcgctc ggtcttgcct tgctcgtcgg tgatgtactt caccagctcc
     4921 gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac gcgcaccccc
     4981 cggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca cgcccatcat
     5041 gaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga ggatcgtggc
     5101 atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag agtttcagca ggccgcccag
     5161 gcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc
     5221 cgtgattttg tagccctggc cgacggccag caggtaggcc gacaggctca tgccggccgc
     5281 cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct tgataggtgg
     5341 gctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat ctgttacgcc
     5401 ggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
     5461 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg
     5521 ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata
     5581 tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta
     5641 tgcagcggaa aagcgctgct tccctgctgt tttgtggaat atctaccgac tggaaacagg
     5701 caaatgcagg aaattactga actgagggga caggcgagag acgatgccaa agagctacac
     5761 cgacgagctg gccgagtggg ttgaatcccg cgcggccaag aagcgccggc gtgatgaggc
     5821 tgcggttgcg ttcctggcgg tgagggcgga tgtcgaggcg gcgttagcgt ccggctatgc
     5881 gctcgtcacc atttgggagc acatgcggga aacggggaag gtcaagttct cctacgagac
     5941 gttccgctcg cacgccaggc ggcacatcaa ggccaagccc gccgatgtgc ccgcaccgca
     6001 ggccaaggct gcggaacccg cgccggcacc caagacgccg gagccacggc ggccgaagca
     6061 ggggggcaag gctgaaaagc cggcccccgc tgcggccccg accggcttca ccttcaaccc
     6121 aacaccggac aaaaaggctg ctaacaaagc tttaaaatcc tctaacgaca tcataacaac
     6181 tgcttctttt tttgaacctc tagttacaat cataggacta tgattttttg ttactctatc
     6241 cattgaatca gatagttcat ttctaaaagt tgaataatta acggtttgca taatctaaat
     6301 ccatgtactt aaataagtac ttaaagtata gtttctagga tttaaaaagt caaaaaagtc
     6361 tttgagtctg tgggattttt ttaaaatccc ctaagatatt ctctttatat attctcttta
     6421 tatattgtca gatttttttt t
//
