LOCUS       sequence-467990-        6474 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 3881 to 3915)
  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 6474)
  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: pKEK3235
FEATURES             Location/Qualifiers
     source          1..6474
                     /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"
     CDS             complement(3133..3156)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(3157..3204)
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     RBS             3857..3868
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(3881..3915)
                     /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(3922..3938)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      3992..4537
                     /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     4438..4457
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4654..4671
                     /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 cacttgtcgt cgtcgtcctt gtagtcggta atgcccgccg cgttcacata
     3181 ttcatgcagg accatgtgat cgcgtttttc gttcggatct ttcgacagca cggtctgggt
     3241 gctcagatag tggttatccg gcagcagaac cgggccatcg ccaatcgggg tgttctgctg
     3301 atagtgatcc gccagctgca ctgagccatc ttccacgtta tggcgcacgg taaagttcgc
     3361 tttaatgcca tttttctgtt tatccgccgt aatgtacacg ttatggctgt taaagttgta
     3421 ttccagtttg tggcccagaa tgttaccatc ttctttaaaa tccgtgcctt tcagttcaat
     3481 gcgattcacc agggtatcgc cttcgaattt cacaacggca cgggttttgt atttgccatc
     3541 atctttgaaa ctaatggtgc gttcctgcac atagccttcc ggcatcgcgc ttttaaaaaa
     3601 atcatggcgt ttcatgtgat ccgggtagcg gctaaaacac tgcacgccgt aggtcagggt
     3661 ggtcaccagg gtcggccacg gcaccggcag tttaccggtg gtgcagataa atttcagggt
     3721 cagtttgcca atggtcgcat cgccttcacc ttcgccacgc acgctaaatt tatggccatt
     3781 cacatcgcca tccagttcca ccagaatcgg caccacgccg gtaaacagtt cttcgccttt
     3841 gctgcccatg ggactctttc tcctctttaa tcgagaccaa gctagcactg tacctaggac
     3901 tgagctagcc gtcaagaatt cactggccgt cgttttacgg tctcagtcga ctactctaga
     3961 aatattttat ctgattaata agatgatctt cttgagatcg ttttggtctg cgcgtaatct
     4021 cttgctctga aaacgaaaaa accgccttgc agggcggttt ttcgaaggtt ctctgagcta
     4081 ccaactcttt gaaccgaggt aactggcttg gaggagcgca gtcaccaaaa cttgtccttt
     4141 cagtttagcc ttaaccggcg catgacttca agactaactc ctctaaatca attaccagtg
     4201 gctgctgcca gtggtgcttt tgcatgtctt tccgggttgg actcaagacg atagttaccg
     4261 gataaggcgc agcggtcgga ctgaacgggg ggttcgtgca tacagtccag cttggagcga
     4321 actgcctacc cggaactgag tgtcaggcgt ggaatgagac aaacgcggcc ataacagcgg
     4381 aatgacaccg gtaaaccgaa aggcaggaac aggagagcgc acgagggagc cgccaggggg
     4441 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac cactgatttg agcgtcagat
     4501 ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaac ggctttgccg cggccctctc
     4561 acttccctgt taagtatctt cctggcatct tccaggaaat ctccgccccg ttcgtaagcc
     4621 atttccgctc gccgcagtcg aacgaccgag cgtagcgagt cagtgagcga ggaagcggaa
     4681 tatatcctgt atcacatatt ctgctgacgc accggtgcag ccttttttct cctgccacat
     4741 gaagcacttc actgacaccc tcatcagtgc caacatagta agccagtata cactccgcta
     4801 gcgctgatgt ccggcggtgc ttttgccgtt acgcaccacc ccgtcagtag ctgaacagga
     4861 gggacagcta aagctagaaa tgtaccaaat tttacctatt ttaggatact tttcatgaag
     4921 ctcttctatt tttccccagt cctttaatag tctaccttta gagtctcgta aatagttatc
     4981 tttgtgacag gggcctcttt tatctttttt aatgtaacta tatgttattc caacgtcact
     5041 attactatta tccaaatctt ttttagcatg ccagtaagaa ctttcataac ttaactctat
     5101 ctttcgacct ctttgatata caacaataaa gctatagcca gtagtaacaa cctgttttac
     5161 ttttgttaaa tctattaact tcttatttat ttttttatgt ttttttgaaa atttaaatat
     5221 ttctatattc attcctacac ttcctcaaat ccaaatggta gcttatgatt ctcttctggt
     5281 ttcttttcta atttttttat atttgcaata aaaactcttt ttctatcttt gattttttta
     5341 ttgtcccaat tcctccaagt atcatcacaa accctttcaa tatcatgtaa atgatgatgt
     5401 ctaaatattg atctgacata atacagatct aggtctagtt catcacttaa cacaacttct
     5461 ctaagtcttt cagatgcttc gattggtatg taatcctctt tatttttagt atctaaaggc
     5521 ttttgcttaa attcttcttc tgtctctgct accttactaa ctgtaaactt gatatttgta
     5581 atcttacgac catgttttct gtgatgatcc ttgtcatcat aggttacaaa aatatccgat
     5641 aattgattaa tctcttctag tgctggtaat aggaacttat ttttaaaatt tgaatatctg
     5701 ttgctgtaac ttttaggtaa atcaaaatca ttaatcatat catcgacata caatacgcaa
     5761 tcaactatat tagcataccc tgcttgttcg cctaatttgc ttttgagaag taagtataat
     5821 ctgcttgaat acttactttt aaatgaaaat agtaactgtc tttctgcttt agtaaagtac
     5881 tcttgtagtt gtatcatgtg tggcattaat gaccaatgaa actcgcaaat taaagcactg
     5941 cttttagggt ctgcttcaat atatgcaaac cagttagcta tcttcgtttg ttctttattc
     6001 agccatactg gcttagacat tattgagtgc attaattgct tcaatctcac tctgttatgc
     6061 ttaacccctg tagctttttc aagatcagat aggcttatct tatacctgtg aaactcttta
     6121 tcttctcttt taaccattga ggcaactaag aatattaagt tttgttcttc ttttgtaagg
     6181 ctatactttc ctgcaacaag agtattagac atagctattt ctttgccagc atttacattt
     6241 ttaacttctt tcatagaact agagtcatta tctcgatata caaattctat aaaacttcta
     6301 ttagtaaaac aactacttca taaaaaaaag tagttttaac gatacaaaaa gtagttttaa
     6361 attcaaaaag tgatacaaaa agtagtttta aattcaaaaa gtgatacaaa aagtagtttt
     6421 aaatttttta aaaaagtgct tcaaagcctt atgtagcaat acttacagag gatt
//
