LOCUS       sequence-467997-        6429 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 3836 to 3870)
  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 6429)
  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: pKEK3236
FEATURES             Location/Qualifiers
     source          1..6429
                     /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             3812..3823
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(3836..3870)
                     /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(3877..3893)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      3947..4492
                     /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     4393..4412
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4609..4626
                     /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 catgtagagt gacgtgcctc cgcgcgctcg tattgctcaa caatggtgta
     3181 gtcctcgtta tgtgacgtaa tatctaactt aatgtccacg ttataggccc cgggcaattg
     3241 cacgggcttc ttggctttgt aagtagtttt gacttccgcg tcatagtgtc caccatcctt
     3301 gagcttgagt cgttggttga tttcaccctt caacgcaccg tcctctgggt acatccgttc
     3361 cgtactagct tcccatccca tagtcttctt ctgcatcact ggtccatcag aagggaaatt
     3421 aatgccgagc agcttaactt tgtagatgaa ctgaccatct tgcagagaag agtcctgggt
     3481 taccgtgact acaccgccgt cttcaaaatt cattacacgc tcccaggtga atccctctgg
     3541 aaaggaaagt ttcaaatagt ccggaatatc agctggatgc tttacatacg ctttcgaacc
     3601 atacataaac tgaggactta agatatccca cgcgaacggc agtggaccgc ccttagttac
     3661 tttcagcttg gcggtttgag ttccttcgta ggggtggcct tcaccctcac cttcgatctc
     3721 gaactcatga ccgttgacag acccttccat gtggacctta aaacgcataa attccttgat
     3781 aattgccata ttatcttctt ccatgggact ctttctcctc tttaatcgag accaagctag
     3841 cactgtacct aggactgagc tagccgtcaa gaattcactg gccgtcgttt tacggtctca
     3901 gtcgactact ctagaaatat tttatctgat taataagatg atcttcttga gatcgttttg
     3961 gtctgcgcgt aatctcttgc tctgaaaacg aaaaaaccgc cttgcagggc ggtttttcga
     4021 aggttctctg agctaccaac tctttgaacc gaggtaactg gcttggagga gcgcagtcac
     4081 caaaacttgt cctttcagtt tagccttaac cggcgcatga cttcaagact aactcctcta
     4141 aatcaattac cagtggctgc tgccagtggt gcttttgcat gtctttccgg gttggactca
     4201 agacgatagt taccggataa ggcgcagcgg tcggactgaa cggggggttc gtgcatacag
     4261 tccagcttgg agcgaactgc ctacccggaa ctgagtgtca ggcgtggaat gagacaaacg
     4321 cggccataac agcggaatga caccggtaaa ccgaaaggca ggaacaggag agcgcacgag
     4381 ggagccgcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccaccactg
     4441 atttgagcgt cagatttcgt gatgcttgtc aggggggcgg agcctatgga aaaacggctt
     4501 tgccgcggcc ctctcacttc cctgttaagt atcttcctgg catcttccag gaaatctccg
     4561 ccccgttcgt aagccatttc cgctcgccgc agtcgaacga ccgagcgtag cgagtcagtg
     4621 agcgaggaag cggaatatat cctgtatcac atattctgct gacgcaccgg tgcagccttt
     4681 tttctcctgc cacatgaagc acttcactga caccctcatc agtgccaaca tagtaagcca
     4741 gtatacactc cgctagcgct gatgtccggc ggtgcttttg ccgttacgca ccaccccgtc
     4801 agtagctgaa caggagggac agctaaagct agaaatgtac caaattttac ctattttagg
     4861 atacttttca tgaagctctt ctatttttcc ccagtccttt aatagtctac ctttagagtc
     4921 tcgtaaatag ttatctttgt gacaggggcc tcttttatct tttttaatgt aactatatgt
     4981 tattccaacg tcactattac tattatccaa atctttttta gcatgccagt aagaactttc
     5041 ataacttaac tctatctttc gacctctttg atatacaaca ataaagctat agccagtagt
     5101 aacaacctgt tttacttttg ttaaatctat taacttctta tttatttttt tatgtttttt
     5161 tgaaaattta aatatttcta tattcattcc tacacttcct caaatccaaa tggtagctta
     5221 tgattctctt ctggtttctt ttctaatttt tttatatttg caataaaaac tctttttcta
     5281 tctttgattt ttttattgtc ccaattcctc caagtatcat cacaaaccct ttcaatatca
     5341 tgtaaatgat gatgtctaaa tattgatctg acataataca gatctaggtc tagttcatca
     5401 cttaacacaa cttctctaag tctttcagat gcttcgattg gtatgtaatc ctctttattt
     5461 ttagtatcta aaggcttttg cttaaattct tcttctgtct ctgctacctt actaactgta
     5521 aacttgatat ttgtaatctt acgaccatgt tttctgtgat gatccttgtc atcataggtt
     5581 acaaaaatat ccgataattg attaatctct tctagtgctg gtaataggaa cttattttta
     5641 aaatttgaat atctgttgct gtaactttta ggtaaatcaa aatcattaat catatcatcg
     5701 acatacaata cgcaatcaac tatattagca taccctgctt gttcgcctaa tttgcttttg
     5761 agaagtaagt ataatctgct tgaatactta cttttaaatg aaaatagtaa ctgtctttct
     5821 gctttagtaa agtactcttg tagttgtatc atgtgtggca ttaatgacca atgaaactcg
     5881 caaattaaag cactgctttt agggtctgct tcaatatatg caaaccagtt agctatcttc
     5941 gtttgttctt tattcagcca tactggctta gacattattg agtgcattaa ttgcttcaat
     6001 ctcactctgt tatgcttaac ccctgtagct ttttcaagat cagataggct tatcttatac
     6061 ctgtgaaact ctttatcttc tcttttaacc attgaggcaa ctaagaatat taagttttgt
     6121 tcttcttttg taaggctata ctttcctgca acaagagtat tagacatagc tatttctttg
     6181 ccagcattta catttttaac ttctttcata gaactagagt cattatctcg atatacaaat
     6241 tctataaaac ttctattagt aaaacaacta cttcataaaa aaaagtagtt ttaacgatac
     6301 aaaaagtagt tttaaattca aaaagtgata caaaaagtag ttttaaattc aaaaagtgat
     6361 acaaaaagta gttttaaatt ttttaaaaaa gtgcttcaaa gccttatgta gcaatactta
     6421 cagaggatt
//
