LOCUS       sequence-471768-        6477 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 6367 to 6401)
  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 6477)
  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: pKEK3230
FEATURES             Location/Qualifiers
     source          1..6477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..546
                     /direction=RIGHT
                     /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     447..466
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     663..680
                     /label=L4440
                     /note="L4440 vector, forward primer"
     oriT            3407..3516
                     /note="incP origin of transfer"
     CDS             3549..3920
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     CDS             complement(4535..5350)
                     /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             complement(4535..5350)
                     /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     5258..5277
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5542..5558
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5542..5558
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     RBS             6343..6354
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(6367..6401)
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     primer_bind     complement(6408..6424)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa ccgccttgca
       61 gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta actggcttgg
      121 aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taaccggcgc atgacttcaa
      181 gactaactcc tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt gcatgtcttt
      241 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcggac tgaacggggg
      301 gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc ggaactgagt gtcaggcgtg
      361 gaatgagaca aacgcggcca taacagcgga atgacaccgg taaaccgaaa ggcaggaaca
      421 ggagagcgca cgagggagcc gccaggggga aacgcctggt atctttatag tcctgtcggg
      481 tttcgccacc actgatttga gcgtcagatt tcgtgatgct tgtcaggggg gcggagccta
      541 tggaaaaacg gctttgccgc ggccctctca cttccctgtt aagtatcttc ctggcatctt
      601 ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga acgaccgagc
      661 gtagcgagtc agtgagcgag gaagcggaat atatcctgta tcacatattc tgctgacgca
      721 ccggtgcagc cttttttctc ctgccacatg aagcacttca ctgacaccct catcagtgcc
      781 aacatagtaa gccagtatac actccgctag cgctgatgtc cggcggtgct tttgccgtta
      841 cgcaccaccc cgtcagtagc tgaacaggag ggacagctaa agctagaaat gtaccaaatt
      901 ttacctattt taggatactt ttcatgaagc tcttctattt ttccccagtc ctttaatagt
      961 ctacctttag agtctcgtaa atagttatct ttgtgacagg ggcctctttt atctttttta
     1021 atgtaactat atgttattcc aacgtcacta ttactattat ccaaatcttt tttagcatgc
     1081 cagtaagaac tttcataact taactctatc tttcgacctc tttgatatac aacaataaag
     1141 ctatagccag tagtaacaac ctgttttact tttgttaaat ctattaactt cttatttatt
     1201 tttttatgtt tttttgaaaa tttaaatatt tctatattca ttcctacact tcctcaaatc
     1261 caaatggtag cttatgattc tcttctggtt tcttttctaa tttttttata tttgcaataa
     1321 aaactctttt tctatctttg atttttttat tgtcccaatt cctccaagta tcatcacaaa
     1381 ccctttcaat atcatgtaaa tgatgatgtc taaatattga tctgacataa tacagatcta
     1441 ggtctagttc atcacttaac acaacttctc taagtctttc agatgcttcg attggtatgt
     1501 aatcctcttt atttttagta tctaaaggct tttgcttaaa ttcttcttct gtctctgcta
     1561 ccttactaac tgtaaacttg atatttgtaa tcttacgacc atgttttctg tgatgatcct
     1621 tgtcatcata ggttacaaaa atatccgata attgattaat ctcttctagt gctggtaata
     1681 ggaacttatt tttaaaattt gaatatctgt tgctgtaact tttaggtaaa tcaaaatcat
     1741 taatcatatc atcgacatac aatacgcaat caactatatt agcataccct gcttgttcgc
     1801 ctaatttgct tttgagaagt aagtataatc tgcttgaata cttactttta aatgaaaata
     1861 gtaactgtct ttctgcttta gtaaagtact cttgtagttg tatcatgtgt ggcattaatg
     1921 accaatgaaa ctcgcaaatt aaagcactgc ttttagggtc tgcttcaata tatgcaaacc
     1981 agttagctat cttcgtttgt tctttattca gccatactgg cttagacatt attgagtgca
     2041 ttaattgctt caatctcact ctgttatgct taacccctgt agctttttca agatcagata
     2101 ggcttatctt atacctgtga aactctttat cttctctttt aaccattgag gcaactaaga
     2161 atattaagtt ttgttcttct tttgtaaggc tatactttcc tgcaacaaga gtattagaca
     2221 tagctatttc tttgccagca tttacatttt taacttcttt catagaacta gagtcattat
     2281 ctcgatatac aaattctata aaacttctat tagtaaaaca actacttcat aaaaaaaagt
     2341 agttttaacg atacaaaaag tagttttaaa ttcaaaaagt gatacaaaaa gtagttttaa
     2401 attcaaaaag tgatacaaaa agtagtttta aattttttaa aaaagtgctt caaagcctta
     2461 tgtagcaata cttacagagg attaaaaaaa aatctgacaa tatataaaga gaatatataa
     2521 agagaatatc ttaggggatt ttaaaaaaat cccacagact caaagacttt tttgactttt
     2581 taaatcctag aaactatact ttaagtactt atttaagtac atggatttag attatgcaaa
     2641 ccgttaatta ttcaactttt agaaatgaac tatctgattc aatggataga gtaacaaaaa
     2701 atcatagtcc tatgattgta actagaggtt caaaaaaaga agcagttgtt atgatgtcgt
     2761 tagaggattt taaagctttg ttagcagcct ttttgtccgg tgttgggttg aaggtgaagc
     2821 cggtcggggc cgcagcgggg gccggctttt cagccttgcc cccctgcttc ggccgccgtg
     2881 gctccggcgt cttgggtgcc ggcgcgggtt ccgcagcctt ggcctgcggt gcgggcacat
     2941 cggcgggctt ggccttgatg tgccgcctgg cgtgcgagcg gaacgtctcg taggagaact
     3001 tgaccttccc cgtttcccgc atgtgctccc aaatggtgac gagcgcatag ccggacgcta
     3061 acgccgcctc gacatccgcc ctcaccgcca ggaacgcaac cgcagcctca tcacgccggc
     3121 gcttcttggc cgcgcgggat tcaacccact cggccagctc gtcggtgtag ctctttggca
     3181 tcgtctctcg cctgtcccct cagttcagta atttcctgca tttgcctgtt tccagtcggt
     3241 agatattcca caaaacagca gggaagcagc gcttttccgc tgcataaccc tgcttcgggg
     3301 tcattatagc gattttttcg gtatatccat cctttttcgc acgatataca ggattttgcc
     3361 aaagggttcg tgtagacttt ccttggtgta tccaacggcg tcagccgggc aggataggtg
     3421 aagtaggccc acccgcgagc gggtgttcct tcttcactgt cccttattcg cacctggcgg
     3481 tgctcaacgg gaatcctgct ctgcgaggct ggccggctac cgccggcgta acagatgagg
     3541 gcaagcggat ggctgatgaa accaagccaa ccaggaaggg cagcccacct atcaaggtgt
     3601 actgccttcc agacgaacga agagcgattg aggaaaaggc ggcggcggcc ggcatgagcc
     3661 tgtcggccta cctgctggcc gtcggccagg gctacaaaat cacgggcgtc gtggactatg
     3721 agcacgtccg cgagctggcc cgcatcaatg gcgacctggg ccgcctgggc ggcctgctga
     3781 aactctggct caccgacgac ccgcgcacgg cgcggttcgg tgatgccacg atcctcgccc
     3841 tgctggcgaa gatcgaagag aagcaggacg agcttggcaa ggtcatgatg ggcgtggtcc
     3901 gcccgagggc agagccatga cttttttagc cgctaaaacg gccggggggt gcgcgtgatt
     3961 gccaagcacg tccccatgcg ctccatcaag aagagcgact tcgcggagct ggtgaagtac
     4021 atcaccgacg agcaaggcaa gaccgagcgc ctgggtcacg tgcgcgtcac gaactgcgag
     4081 gcaaacaccc tgcccgctgt catggccgag gtgatggcga cccagcacgg caacacccgt
     4141 tccgaggccg acaagaccta tcacctcctg gttagcttcc gcgcgggaga gaagcccgac
     4201 gcggagacgt tgcgcgcgat tgaggaccgc atctgcgctg ggcttggctt cgccgagcat
     4261 cagcgcgtca gtgccgtgca tcacgacacc gacaacctgc acatccatat cgccatcaac
     4321 aagattcacc cgacccgaaa caccatccat gagccgtatc gggcctaccg cgccctcgct
     4381 gacctctgcg cgacgctcga acgggactac gggcttgagc gtgacaatca cgaaacgcgg
     4441 cagcgcgttt ccgagaaccg cgcgaacgac atggagcggc acgcgggcgt ggaaagcctg
     4501 gtcggctgct cgcgctttgt tagcagccgg atccttagaa aaactcatcg agcatcaaat
     4561 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     4621 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     4681 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     4741 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagct
     4801 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     4861 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     4921 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     4981 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     5041 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     5101 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     5161 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     5221 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     5281 ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt tcccgttgaa
     5341 tatggctcat atgtttagtc tccttttttt gttataaata ttttatacac aaaaatcttc
     5401 gttatttaaa taagttttat agctattttt tcaagataca aaactattaa ttagttataa
     5461 ttaggttatt agcttaaaat tataaaccaa atacgatgag tgacaaccca aaagatctcg
     5521 atcctctacg ccgttgggga gcaggaaaca gctatgacgg taccaaaaaa aaccccgccc
     5581 tgtcaggggc ggggtttttt tttgagctct tattagctct tatacagctc gtccataccc
     5641 aggctcaagc ccgcagcggt cacaaattcc accaggacca tgtgatcacg gcgttcctgc
     5701 ggatctttgc tcagcacgct attggtcgtc agatagtgtt gttccggcag caacaccgga
     5761 ccctcaccaa ttggggtgtt ctgttggtaa tggtcggcca gttggacgct accgtcatca
     5821 acattgtggc gaatacggaa ttgggcctcc aagccgttac gattcttgtc tgccgtaatg
     5881 taaacataat ggctgttgaa gctatactcc agtttatggc ccagaatgtt accatcctct
     5941 ttaaactcca aacccttcaa atcgatacga ttgaccagcg cttcaccttc aaacttaacc
     6001 tctgcgcgag ttttgtacgt gccatcttcc ttgaaggaaa tggtacgctc ctggacgtaa
     6061 ccgtccggca tggcactttt aaagaaatcg ttttggcgca tgtgctccgg atacttcgca
     6121 aaacactgca cgccatacga caaggtcgtc accaaggtcg gccaaggcac cggcaggcga
     6181 ccggtcgtgc agatgaactt aatttccagt ttgccgttgc tcgcgtcgcc ataaccttca
     6241 ccgcgaacgc taaaacgatg accgttcacg tcgccctcca gctcgatcag aatcggaacc
     6301 aagcccgaga aaatatcttc gccactgctc accatgggac tctttctcct ctttaatcga
     6361 gaccaagcta gcactgtacc taggactgag ctagccgtca agaattcact ggccgtcgtt
     6421 ttacggtctc agtcgactac tctagaaata ttttatctga ttaataagat gatcttc
//
