LOCUS       sequence-464927-        6486 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 3893 to 3927)
  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 6486)
  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: pKEK3231
FEATURES             Location/Qualifiers
     source          1..6486
                     /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..3150)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3869..3880
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(3893..3927)
                     /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(3934..3950)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      4004..4549
                     /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     4450..4469
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4666..4683
                     /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 cagtgatgat gatgatggtg ctgatatgtg tctaagtcta cggcttttac
     3181 aacttctacg aggcacatgt gatctcttct ctcatctcta tctttagata atttagtgtg
     3241 gtaagtgata tgatgatacc ttggtatatg cacagcagca cttccggcga gaggcctgtt
     3301 catctgggaa cacttggtga ccagtttttc agtcaccccc tcaatatcgt atgcttgatt
     3361 aaattccact cggattccat tgttaacggt atcgggcagt atatacagga tgctgggagg
     3421 acattgaaaa gcaacgttct tcctcagaat gtgtccatcc tttttgaagt tttctccggt
     3481 caaggtcacc ctgttgtata tagaccctct ctcataggtt accatagctc ttgtcttata
     3541 tacgccgtcg ccttcaaaac ttatagttct ttcttgggtg taaccctctg gcattgctga
     3601 cttaaagaaa tcttttatat ggctggggta cttagcaaaa cattggaccc catagctaag
     3661 ggttgacacc agtgtagccc agggtactgg taaatcgcca gttgtacaga tatatttggc
     3721 cttaatagta ccagtggtag cgtcgccagt tccctccccc ttgattatga acttcatgcc
     3781 ctccacatcc ccttcaagtt ctgttatgta gggtatttct ttttcaaaca gtttagcacc
     3841 ctctgtcaag gcagtagcca tgggactctt tctcctcttt aatcgagacc aagctagcac
     3901 tgtacctagg actgagctag ccgtcaagaa ttcactggcc gtcgttttac ggtctcagtc
     3961 gactactcta gaaatatttt atctgattaa taagatgatc ttcttgagat cgttttggtc
     4021 tgcgcgtaat ctcttgctct gaaaacgaaa aaaccgcctt gcagggcggt ttttcgaagg
     4081 ttctctgagc taccaactct ttgaaccgag gtaactggct tggaggagcg cagtcaccaa
     4141 aacttgtcct ttcagtttag ccttaaccgg cgcatgactt caagactaac tcctctaaat
     4201 caattaccag tggctgctgc cagtggtgct tttgcatgtc tttccgggtt ggactcaaga
     4261 cgatagttac cggataaggc gcagcggtcg gactgaacgg ggggttcgtg catacagtcc
     4321 agcttggagc gaactgccta cccggaactg agtgtcaggc gtggaatgag acaaacgcgg
     4381 ccataacagc ggaatgacac cggtaaaccg aaaggcagga acaggagagc gcacgaggga
     4441 gccgccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc accactgatt
     4501 tgagcgtcag atttcgtgat gcttgtcagg ggggcggagc ctatggaaaa acggctttgc
     4561 cgcggccctc tcacttccct gttaagtatc ttcctggcat cttccaggaa atctccgccc
     4621 cgttcgtaag ccatttccgc tcgccgcagt cgaacgaccg agcgtagcga gtcagtgagc
     4681 gaggaagcgg aatatatcct gtatcacata ttctgctgac gcaccggtgc agcctttttt
     4741 ctcctgccac atgaagcact tcactgacac cctcatcagt gccaacatag taagccagta
     4801 tacactccgc tagcgctgat gtccggcggt gcttttgccg ttacgcacca ccccgtcagt
     4861 agctgaacag gagggacagc taaagctaga aatgtaccaa attttaccta ttttaggata
     4921 cttttcatga agctcttcta tttttcccca gtcctttaat agtctacctt tagagtctcg
     4981 taaatagtta tctttgtgac aggggcctct tttatctttt ttaatgtaac tatatgttat
     5041 tccaacgtca ctattactat tatccaaatc ttttttagca tgccagtaag aactttcata
     5101 acttaactct atctttcgac ctctttgata tacaacaata aagctatagc cagtagtaac
     5161 aacctgtttt acttttgtta aatctattaa cttcttattt atttttttat gtttttttga
     5221 aaatttaaat atttctatat tcattcctac acttcctcaa atccaaatgg tagcttatga
     5281 ttctcttctg gtttcttttc taattttttt atatttgcaa taaaaactct ttttctatct
     5341 ttgatttttt tattgtccca attcctccaa gtatcatcac aaaccctttc aatatcatgt
     5401 aaatgatgat gtctaaatat tgatctgaca taatacagat ctaggtctag ttcatcactt
     5461 aacacaactt ctctaagtct ttcagatgct tcgattggta tgtaatcctc tttattttta
     5521 gtatctaaag gcttttgctt aaattcttct tctgtctctg ctaccttact aactgtaaac
     5581 ttgatatttg taatcttacg accatgtttt ctgtgatgat ccttgtcatc ataggttaca
     5641 aaaatatccg ataattgatt aatctcttct agtgctggta ataggaactt atttttaaaa
     5701 tttgaatatc tgttgctgta acttttaggt aaatcaaaat cattaatcat atcatcgaca
     5761 tacaatacgc aatcaactat attagcatac cctgcttgtt cgcctaattt gcttttgaga
     5821 agtaagtata atctgcttga atacttactt ttaaatgaaa atagtaactg tctttctgct
     5881 ttagtaaagt actcttgtag ttgtatcatg tgtggcatta atgaccaatg aaactcgcaa
     5941 attaaagcac tgcttttagg gtctgcttca atatatgcaa accagttagc tatcttcgtt
     6001 tgttctttat tcagccatac tggcttagac attattgagt gcattaattg cttcaatctc
     6061 actctgttat gcttaacccc tgtagctttt tcaagatcag ataggcttat cttatacctg
     6121 tgaaactctt tatcttctct tttaaccatt gaggcaacta agaatattaa gttttgttct
     6181 tcttttgtaa ggctatactt tcctgcaaca agagtattag acatagctat ttctttgcca
     6241 gcatttacat ttttaacttc tttcatagaa ctagagtcat tatctcgata tacaaattct
     6301 ataaaacttc tattagtaaa acaactactt cataaaaaaa agtagtttta acgatacaaa
     6361 aagtagtttt aaattcaaaa agtgatacaa aaagtagttt taaattcaaa aagtgataca
     6421 aaaagtagtt ttaaattttt taaaaaagtg cttcaaagcc ttatgtagca atacttacag
     6481 aggatt
//
