LOCUS       sequence-467985-        6458 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 6458)
  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: pKEK3234
FEATURES             Location/Qualifiers
     source          1..6458
                     /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)"
     CDS             3313..3330
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3384..3400)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3384..3400)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3592..4407
                     /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             3592..4407
                     /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(3665..3684)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(5022..5393)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            5426..5535
                     /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 cagcctagta
     2641 aagaaggata tgtgcatcaa aatgactatg gagggaaccg ttaatggtca ccacttcaaa
     2701 tgcgtaggcg aaggtgaggg taagccattt gagggtactc aggtcgagaa gatcagaatc
     2761 actgagggtg gacctctccc atttgcttac gacattctag ctccatgttg tatgtatggc
     2821 tcaaaaactt tcatcaagca cgtaagtggt attcccgatt atttcaaaga aagctttcca
     2881 gagggcttca cttgggaaag gactcagatt tttgaggacg gtggttatct caccatacat
     2941 caggatacct ctctacaggg caacaatttt atcttcaaag taaatgtgat cggcgctaat
     3001 ttccccgcca atgggccagt tatgcagaaa aagacagctg gctgggaacc atgcgtggag
     3061 atgctctacc ccagggacgg agtactttgc ggtcagagtc taatggcctt aaaatgtacc
     3121 gacgggaacc acctaacttc acacttaaga actacctaca ggagccgaaa gccatccaac
     3181 gctgttaata tgccagaatt tcatttcggg gaccatcgta tagaaattct aaaggccgaa
     3241 caagggaagt tctacgaaca atatgaatca gccgtggcca ggtactgcga ggccgcacct
     3301 tctaagcttg gccaccatca ccatcaccat taagagctca aaaaaaaccc cgcccctgac
     3361 agggcggggt tttttttggt accgtcatag ctgtttcctg ctccccaacg gcgtagagga
     3421 tcgagatctt ttgggttgtc actcatcgta tttggtttat aattttaagc taataaccta
     3481 attataacta attaatagtt ttgtatcttg aaaaaatagc tataaaactt atttaaataa
     3541 cgaagatttt tgtgtataaa atatttataa caaaaaaagg agactaaaca tatgagccat
     3601 attcaacggg aaacgtcttg ctcgaggccg cgattaaatt ccaacatgga tgctgattta
     3661 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
     3721 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     3781 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     3841 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg
     3901 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     3961 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     4021 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     4081 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     4141 aagcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     4201 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     4261 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     4321 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     4381 catttgatgc tcgatgagtt tttctaagga tccggctgct aacaaagcgc gagcagccga
     4441 ccaggctttc cacgcccgcg tgccgctcca tgtcgttcgc gcggttctcg gaaacgcgct
     4501 gccgcgtttc gtgattgtca cgctcaagcc cgtagtcccg ttcgagcgtc gcgcagaggt
     4561 cagcgagggc gcggtaggcc cgatacggct catggatggt gtttcgggtc gggtgaatct
     4621 tgttgatggc gatatggatg tgcaggttgt cggtgtcgtg atgcacggca ctgacgcgct
     4681 gatgctcggc gaagccaagc ccagcgcaga tgcggtcctc aatcgcgcgc aacgtctccg
     4741 cgtcgggctt ctctcccgcg cggaagctaa ccaggaggtg ataggtcttg tcggcctcgg
     4801 aacgggtgtt gccgtgctgg gtcgccatca cctcggccat gacagcgggc agggtgtttg
     4861 cctcgcagtt cgtgacgcgc acgtgaccca ggcgctcggt cttgccttgc tcgtcggtga
     4921 tgtacttcac cagctccgcg aagtcgctct tcttgatgga gcgcatgggg acgtgcttgg
     4981 caatcacgcg caccccccgg ccgttttagc ggctaaaaaa gtcatggctc tgccctcggg
     5041 cggaccacgc ccatcatgac cttgccaagc tcgtcctgct tctcttcgat cttcgccagc
     5101 agggcgagga tcgtggcatc accgaaccgc gccgtgcgcg ggtcgtcggt gagccagagt
     5161 ttcagcaggc cgcccaggcg gcccaggtcg ccattgatgc gggccagctc gcggacgtgc
     5221 tcatagtcca cgacgcccgt gattttgtag ccctggccga cggccagcag gtaggccgac
     5281 aggctcatgc cggccgccgc cgccttttcc tcaatcgctc ttcgttcgtc tggaaggcag
     5341 tacaccttga taggtgggct gcccttcctg gttggcttgg tttcatcagc catccgcttg
     5401 ccctcatctg ttacgccggc ggtagccggc cagcctcgca gagcaggatt cccgttgagc
     5461 accgccaggt gcgaataagg gacagtgaag aaggaacacc cgctcgcggg tgggcctact
     5521 tcacctatcc tgcccggctg acgccgttgg atacaccaag gaaagtctac acgaaccctt
     5581 tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata ccgaaaaaat cgctataatg
     5641 accccgaagc agggttatgc agcggaaaag cgctgcttcc ctgctgtttt gtggaatatc
     5701 taccgactgg aaacaggcaa atgcaggaaa ttactgaact gaggggacag gcgagagacg
     5761 atgccaaaga gctacaccga cgagctggcc gagtgggttg aatcccgcgc ggccaagaag
     5821 cgccggcgtg atgaggctgc ggttgcgttc ctggcggtga gggcggatgt cgaggcggcg
     5881 ttagcgtccg gctatgcgct cgtcaccatt tgggagcaca tgcgggaaac ggggaaggtc
     5941 aagttctcct acgagacgtt ccgctcgcac gccaggcggc acatcaaggc caagcccgcc
     6001 gatgtgcccg caccgcaggc caaggctgcg gaacccgcgc cggcacccaa gacgccggag
     6061 ccacggcggc cgaagcaggg gggcaaggct gaaaagccgg cccccgctgc ggccccgacc
     6121 ggcttcacct tcaacccaac accggacaaa aaggctgcta acaaagcttt aaaatcctct
     6181 aacgacatca taacaactgc ttcttttttt gaacctctag ttacaatcat aggactatga
     6241 ttttttgtta ctctatccat tgaatcagat agttcatttc taaaagttga ataattaacg
     6301 gtttgcataa tctaaatcca tgtacttaaa taagtactta aagtatagtt tctaggattt
     6361 aaaaagtcaa aaaagtcttt gagtctgtgg gattttttta aaatccccta agatattctc
     6421 tttatatatt ctctttatat attgtcagat tttttttt
//
