LOCUS       sequence-464917-        6438 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 3845 to 3879)
  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 6438)
  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: pKEK3229
FEATURES             Location/Qualifiers
     source          1..6438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            924..1033
                     /note="incP origin of transfer"
     CDS             1066..1437
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     CDS             complement(2052..2867)
                     /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(2052..2867)
                     /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     2775..2794
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3059..3075
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3059..3075
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     RBS             3821..3832
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(3845..3879)
                     /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(3886..3902)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      3956..4501
                     /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     4402..4421
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4618..4635
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaaaaaaat ctgacaatat ataaagagaa tatataaaga gaatatctta ggggatttta
       61 aaaaaatccc acagactcaa agactttttt gactttttaa atcctagaaa ctatacttta
      121 agtacttatt taagtacatg gatttagatt atgcaaaccg ttaattattc aacttttaga
      181 aatgaactat ctgattcaat ggatagagta acaaaaaatc atagtcctat gattgtaact
      241 agaggttcaa aaaaagaagc agttgttatg atgtcgttag aggattttaa agctttgtta
      301 gcagcctttt tgtccggtgt tgggttgaag gtgaagccgg tcggggccgc agcgggggcc
      361 ggcttttcag ccttgccccc ctgcttcggc cgccgtggct ccggcgtctt gggtgccggc
      421 gcgggttccg cagccttggc ctgcggtgcg ggcacatcgg cgggcttggc cttgatgtgc
      481 cgcctggcgt gcgagcggaa cgtctcgtag gagaacttga ccttccccgt ttcccgcatg
      541 tgctcccaaa tggtgacgag cgcatagccg gacgctaacg ccgcctcgac atccgccctc
      601 accgccagga acgcaaccgc agcctcatca cgccggcgct tcttggccgc gcgggattca
      661 acccactcgg ccagctcgtc ggtgtagctc tttggcatcg tctctcgcct gtcccctcag
      721 ttcagtaatt tcctgcattt gcctgtttcc agtcggtaga tattccacaa aacagcaggg
      781 aagcagcgct tttccgctgc ataaccctgc ttcggggtca ttatagcgat tttttcggta
      841 tatccatcct ttttcgcacg atatacagga ttttgccaaa gggttcgtgt agactttcct
      901 tggtgtatcc aacggcgtca gccgggcagg ataggtgaag taggcccacc cgcgagcggg
      961 tgttccttct tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa tcctgctctg
     1021 cgaggctggc cggctaccgc cggcgtaaca gatgagggca agcggatggc tgatgaaacc
     1081 aagccaacca ggaagggcag cccacctatc aaggtgtact gccttccaga cgaacgaaga
     1141 gcgattgagg aaaaggcggc ggcggccggc atgagcctgt cggcctacct gctggccgtc
     1201 ggccagggct acaaaatcac gggcgtcgtg gactatgagc acgtccgcga gctggcccgc
     1261 atcaatggcg acctgggccg cctgggcggc ctgctgaaac tctggctcac cgacgacccg
     1321 cgcacggcgc ggttcggtga tgccacgatc ctcgccctgc tggcgaagat cgaagagaag
     1381 caggacgagc ttggcaaggt catgatgggc gtggtccgcc cgagggcaga gccatgactt
     1441 ttttagccgc taaaacggcc ggggggtgcg cgtgattgcc aagcacgtcc ccatgcgctc
     1501 catcaagaag agcgacttcg cggagctggt gaagtacatc accgacgagc aaggcaagac
     1561 cgagcgcctg ggtcacgtgc gcgtcacgaa ctgcgaggca aacaccctgc ccgctgtcat
     1621 ggccgaggtg atggcgaccc agcacggcaa cacccgttcc gaggccgaca agacctatca
     1681 cctcctggtt agcttccgcg cgggagagaa gcccgacgcg gagacgttgc gcgcgattga
     1741 ggaccgcatc tgcgctgggc ttggcttcgc cgagcatcag cgcgtcagtg ccgtgcatca
     1801 cgacaccgac aacctgcaca tccatatcgc catcaacaag attcacccga cccgaaacac
     1861 catccatgag ccgtatcggg cctaccgcgc cctcgctgac ctctgcgcga cgctcgaacg
     1921 ggactacggg cttgagcgtg acaatcacga aacgcggcag cgcgtttccg agaaccgcgc
     1981 gaacgacatg gagcggcacg cgggcgtgga aagcctggtc ggctgctcgc gctttgttag
     2041 cagccggatc cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     2101 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     2161 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     2221 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     2281 tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt
     2341 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2401 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     2461 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     2521 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     2581 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     2641 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     2701 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     2761 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     2821 tttaatcgcg gcctcgagca agacgtttcc cgttgaatat ggctcatatg tttagtctcc
     2881 tttttttgtt ataaatattt tatacacaaa aatcttcgtt atttaaataa gttttatagc
     2941 tattttttca agatacaaaa ctattaatta gttataatta ggttattagc ttaaaattat
     3001 aaaccaaata cgatgagtga caacccaaaa gatctcgatc ctctacgccg ttggggagca
     3061 ggaaacagct atgacggtac caaaaaaaac cccgccctgt caggggcggg gttttttttt
     3121 gagctcttat tatgggagag ccttcggcag cggtgagaaa tggccttcgg ctgcctcctg
     3181 ctggtcccaa gttgtgaaat ctttatcgtg tttggtcagt tccaggcggt gttcaacgaa
     3241 atgaaattct ggcagcttga caggtttttt ggctttatac gtcgttttga aatcaacacg
     3301 atgatgaccg cctcctttca gtttaaaaac cattgcagtg tcacttttta acatcccgtc
     3361 acgcacgtac agtgtttcca cgctcggctc ccatccagca atggtttttt gcattacggg
     3421 cccatccgga ggcaggttca caccatgaaa tttaacatca tagatgaaac agttctcttg
     3481 caagtttatg ctggagtgat tactggcggt gcatacgcca ccatcttcaa aaatcatcga
     3541 ccgctcccaa gacatacctt cgggaaaaga ctgtttgtgg tagtctggga tatccgctgg
     3601 atacttatta aacggtttgc ttccatacat gtggctaggt gtaagaatat gaacggaaaa
     3661 cggcagaggc gcgcctttgg tgactgtcat tttcatgtgc tgtaaccctt cgtacggctt
     3721 gccttcacct ttaccctcga tcgtaaaatc atggccattg acagtgccct caaggtgcaa
     3781 cttggtcttc attacctgct taatcactgc catgggactc tttctcctct ttaatcgaga
     3841 ccaagctagc actgtaccta ggactgagct agccgtcaag aattcactgg ccgtcgtttt
     3901 acggtctcag tcgactactc tagaaatatt ttatctgatt aataagatga tcttcttgag
     3961 atcgttttgg tctgcgcgta atctcttgct ctgaaaacga aaaaaccgcc ttgcagggcg
     4021 gtttttcgaa ggttctctga gctaccaact ctttgaaccg aggtaactgg cttggaggag
     4081 cgcagtcacc aaaacttgtc ctttcagttt agccttaacc ggcgcatgac ttcaagacta
     4141 actcctctaa atcaattacc agtggctgct gccagtggtg cttttgcatg tctttccggg
     4201 ttggactcaa gacgatagtt accggataag gcgcagcggt cggactgaac ggggggttcg
     4261 tgcatacagt ccagcttgga gcgaactgcc tacccggaac tgagtgtcag gcgtggaatg
     4321 agacaaacgc ggccataaca gcggaatgac accggtaaac cgaaaggcag gaacaggaga
     4381 gcgcacgagg gagccgccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4441 ccaccactga tttgagcgtc agatttcgtg atgcttgtca ggggggcgga gcctatggaa
     4501 aaacggcttt gccgcggccc tctcacttcc ctgttaagta tcttcctggc atcttccagg
     4561 aaatctccgc cccgttcgta agccatttcc gctcgccgca gtcgaacgac cgagcgtagc
     4621 gagtcagtga gcgaggaagc ggaatatatc ctgtatcaca tattctgctg acgcaccggt
     4681 gcagcctttt ttctcctgcc acatgaagca cttcactgac accctcatca gtgccaacat
     4741 agtaagccag tatacactcc gctagcgctg atgtccggcg gtgcttttgc cgttacgcac
     4801 caccccgtca gtagctgaac aggagggaca gctaaagcta gaaatgtacc aaattttacc
     4861 tattttagga tacttttcat gaagctcttc tatttttccc cagtccttta atagtctacc
     4921 tttagagtct cgtaaatagt tatctttgtg acaggggcct cttttatctt ttttaatgta
     4981 actatatgtt attccaacgt cactattact attatccaaa tcttttttag catgccagta
     5041 agaactttca taacttaact ctatctttcg acctctttga tatacaacaa taaagctata
     5101 gccagtagta acaacctgtt ttacttttgt taaatctatt aacttcttat ttattttttt
     5161 atgttttttt gaaaatttaa atatttctat attcattcct acacttcctc aaatccaaat
     5221 ggtagcttat gattctcttc tggtttcttt tctaattttt ttatatttgc aataaaaact
     5281 ctttttctat ctttgatttt tttattgtcc caattcctcc aagtatcatc acaaaccctt
     5341 tcaatatcat gtaaatgatg atgtctaaat attgatctga cataatacag atctaggtct
     5401 agttcatcac ttaacacaac ttctctaagt ctttcagatg cttcgattgg tatgtaatcc
     5461 tctttatttt tagtatctaa aggcttttgc ttaaattctt cttctgtctc tgctacctta
     5521 ctaactgtaa acttgatatt tgtaatctta cgaccatgtt ttctgtgatg atccttgtca
     5581 tcataggtta caaaaatatc cgataattga ttaatctctt ctagtgctgg taataggaac
     5641 ttatttttaa aatttgaata tctgttgctg taacttttag gtaaatcaaa atcattaatc
     5701 atatcatcga catacaatac gcaatcaact atattagcat accctgcttg ttcgcctaat
     5761 ttgcttttga gaagtaagta taatctgctt gaatacttac ttttaaatga aaatagtaac
     5821 tgtctttctg ctttagtaaa gtactcttgt agttgtatca tgtgtggcat taatgaccaa
     5881 tgaaactcgc aaattaaagc actgctttta gggtctgctt caatatatgc aaaccagtta
     5941 gctatcttcg tttgttcttt attcagccat actggcttag acattattga gtgcattaat
     6001 tgcttcaatc tcactctgtt atgcttaacc cctgtagctt tttcaagatc agataggctt
     6061 atcttatacc tgtgaaactc tttatcttct cttttaacca ttgaggcaac taagaatatt
     6121 aagttttgtt cttcttttgt aaggctatac tttcctgcaa caagagtatt agacatagct
     6181 atttctttgc cagcatttac atttttaact tctttcatag aactagagtc attatctcga
     6241 tatacaaatt ctataaaact tctattagta aaacaactac ttcataaaaa aaagtagttt
     6301 taacgataca aaaagtagtt ttaaattcaa aaagtgatac aaaaagtagt tttaaattca
     6361 aaaagtgata caaaaagtag ttttaaattt tttaaaaaag tgcttcaaag ccttatgtag
     6421 caatacttac agaggatt
//
