LOCUS       sequence-464918-        6456 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 6456)
  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: pKEK3233
FEATURES             Location/Qualifiers
     source          1..6456
                     /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             3307..3324
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3382..3398)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3382..3398)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3590..4405
                     /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             3590..4405
                     /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(3663..3682)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(5020..5391)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            5424..5533
                     /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 ttccctgtca
     2641 aaacaagttt tgccaagaga tgtcagaatg cgattccaca tggacggttg cgttaatggt
     2701 catcaattta ccattgaagg tgaaggtgca ggcaaacctt acgagggtaa aaaaaccctg
     2761 aaactcagag tcacaaaggg gggtcctctc ccttttgcct ttgatatact gagtgctacc
     2821 ttcacctatg gcaaccgatg tttttgcgat taccccgaag atatgccaga ttactttaaa
     2881 caaagcctgc ccgagggcta ttcttgggag cgtacattaa tgttcgagga tggcggctgc
     2941 gggaccgcct ctgctcacat aagtctggaa aaagattgtt ttatccacaa ttccaccttt
     3001 cacggagtca acttcccagc taatggacct gtaatgcaga agaagacact taactgggaa
     3061 cccagttcag aacttatcac tgcttgtgat gggatactga agggggacgt taccatgttc
     3121 ttattattgg aaggaggaca tagacttaaa tgccaattta caacatcata caaggcccac
     3181 aaggccgtaa agatgccccc caaccacatt attgaacacg tcctggtgaa gaaagaagta
     3241 gctgatggat ttcaaatcca ggaacacgca gttgctaaac acttcacagt cgatgtcaag
     3301 gaaacacatc accaccatca ccattgataa gagctcaaaa aaaaaccccg cccctgacag
     3361 ggcggggttt tttttggtac cgtcatagct gtttcctgct ccccaacggc gtagaggatc
     3421 gagatctttt gggttgtcac tcatcgtatt tggtttataa ttttaagcta ataacctaat
     3481 tataactaat taatagtttt gtatcttgaa aaaatagcta taaaacttat ttaaataacg
     3541 aagatttttg tgtataaaat atttataaca aaaaaaggag actaaacata tgagccatat
     3601 tcaacgggaa acgtcttgct cgaggccgcg attaaattcc aacatggatg ctgatttata
     3661 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta
     3721 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     3781 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     3841 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa
     3901 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     3961 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     4021 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     4081 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     4141 gcttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     4201 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     4261 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     4321 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     4381 tttgatgctc gatgagtttt tctaaggatc cggctgctaa caaagcgcga gcagccgacc
     4441 aggctttcca cgcccgcgtg ccgctccatg tcgttcgcgc ggttctcgga aacgcgctgc
     4501 cgcgtttcgt gattgtcacg ctcaagcccg tagtcccgtt cgagcgtcgc gcagaggtca
     4561 gcgagggcgc ggtaggcccg atacggctca tggatggtgt ttcgggtcgg gtgaatcttg
     4621 ttgatggcga tatggatgtg caggttgtcg gtgtcgtgat gcacggcact gacgcgctga
     4681 tgctcggcga agccaagccc agcgcagatg cggtcctcaa tcgcgcgcaa cgtctccgcg
     4741 tcgggcttct ctcccgcgcg gaagctaacc aggaggtgat aggtcttgtc ggcctcggaa
     4801 cgggtgttgc cgtgctgggt cgccatcacc tcggccatga cagcgggcag ggtgtttgcc
     4861 tcgcagttcg tgacgcgcac gtgacccagg cgctcggtct tgccttgctc gtcggtgatg
     4921 tacttcacca gctccgcgaa gtcgctcttc ttgatggagc gcatggggac gtgcttggca
     4981 atcacgcgca ccccccggcc gttttagcgg ctaaaaaagt catggctctg ccctcgggcg
     5041 gaccacgccc atcatgacct tgccaagctc gtcctgcttc tcttcgatct tcgccagcag
     5101 ggcgaggatc gtggcatcac cgaaccgcgc cgtgcgcggg tcgtcggtga gccagagttt
     5161 cagcaggccg cccaggcggc ccaggtcgcc attgatgcgg gccagctcgc ggacgtgctc
     5221 atagtccacg acgcccgtga ttttgtagcc ctggccgacg gccagcaggt aggccgacag
     5281 gctcatgccg gccgccgccg ccttttcctc aatcgctctt cgttcgtctg gaaggcagta
     5341 caccttgata ggtgggctgc ccttcctggt tggcttggtt tcatcagcca tccgcttgcc
     5401 ctcatctgtt acgccggcgg tagccggcca gcctcgcaga gcaggattcc cgttgagcac
     5461 cgccaggtgc gaataaggga cagtgaagaa ggaacacccg ctcgcgggtg ggcctacttc
     5521 acctatcctg cccggctgac gccgttggat acaccaagga aagtctacac gaaccctttg
     5581 gcaaaatcct gtatatcgtg cgaaaaagga tggatatacc gaaaaaatcg ctataatgac
     5641 cccgaagcag ggttatgcag cggaaaagcg ctgcttccct gctgttttgt ggaatatcta
     5701 ccgactggaa acaggcaaat gcaggaaatt actgaactga ggggacaggc gagagacgat
     5761 gccaaagagc tacaccgacg agctggccga gtgggttgaa tcccgcgcgg ccaagaagcg
     5821 ccggcgtgat gaggctgcgg ttgcgttcct ggcggtgagg gcggatgtcg aggcggcgtt
     5881 agcgtccggc tatgcgctcg tcaccatttg ggagcacatg cgggaaacgg ggaaggtcaa
     5941 gttctcctac gagacgttcc gctcgcacgc caggcggcac atcaaggcca agcccgccga
     6001 tgtgcccgca ccgcaggcca aggctgcgga acccgcgccg gcacccaaga cgccggagcc
     6061 acggcggccg aagcaggggg gcaaggctga aaagccggcc cccgctgcgg ccccgaccgg
     6121 cttcaccttc aacccaacac cggacaaaaa ggctgctaac aaagctttaa aatcctctaa
     6181 cgacatcata acaactgctt ctttttttga acctctagtt acaatcatag gactatgatt
     6241 ttttgttact ctatccattg aatcagatag ttcatttcta aaagttgaat aattaacggt
     6301 ttgcataatc taaatccatg tacttaaata agtacttaaa gtatagtttc taggatttaa
     6361 aaagtcaaaa aagtctttga gtctgtggga tttttttaaa atcccctaag atattctctt
     6421 tatatattct ctttatatat tgtcagattt tttttt
//
