LOCUS       sequence-460344-        8789 bp    DNA     circular SYN 18-AUG-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 6923 to 6957)
  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 8789)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 18, 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: pKEK3227
FEATURES             Location/Qualifiers
     source          1..8789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      443..837
                     /label=RSF1010 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     RSF1010; requires the RSF1010 RepA/B/C proteins for 
                     replication (Scholz et al., 1989)"
     CDS             2382..3353
                     /codon_start=1
                     /gene="repB"
                     /product="replication protein B of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepB
                     /translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDATTGQMMNREWSAAEVL
                     QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
                     YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
                     EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
     CDS             3864..4703
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein A of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepA
                     /translation="MATHKPINILEAFAAAPPPLDYVLPNMVAGTVGALVSPGGAGKSM
                     LALQLAAQIAGGPDLLEVGELPTGPVIYLPAEDPPTAIHHRLHALGAHLSAEERQAVAD
                     GLLIQPLIGSLPNIMAPEWFDGLKRAAEGRRLMVLDTLRRFHIEEENASGPMAQVIGRM
                     EAIAADTGCSIVFLHHASKGAAMMGAGDQQQASRGSSVLVDNIRWQSYLSSMTSAEAEE
                     WGVDDDQRRFFVRFGVSKANYGAPFADRWFRRHDGGVLKPAVLERQRKSKGVPRGEA"
     CDS             4690..5541
                     /codon_start=1
                     /gene="repC"
                     /product="replication protein C of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepC
                     /translation="MVKPKNKHSLSHVRHDPAHCLAPGLFRALKRGERKRSKLDVTYDY
                     GDGKRIEFSGPEPLGADDLRILQGLVAMAGPNGLVLGPEPKTEGGRQLRLFLEPKWEAV
                     TADAMVVKGSYRALAKEIGAEVDSGGALKHIQDCIERLWKVSIIAQNGRKRQGFRLLSE
                     YASDEADGRLYVALNPLIAQAVMGGGQHVRISMDEVRALDSETARLLHQRLCGWIDPGK
                     TGKASIDTLCGYVWPSEASGSTMRKRRQRVREALPELVALGWTVTEFAAGKYDITRPKA
                     AG"
     primer_bind     complement(6167..6184)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6301..6846)
                     /direction=LEFT
                     /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     complement(6381..6400)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6900..6916
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6923..6957
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     RBS             6970..6981
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             7670..7687
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7745..7761)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7745..7761)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             7953..8768
                     /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             7953..8768
                     /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     complement(8026..8045)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 gcgagcctgt cagaccaagt ttactcatat atactttaga ttgatttctg aaagcgacca
       61 ggtgctcggc gtggcaagac tcgcagcgaa cccgtagaaa gccatgctcc agccgcccgc
      121 attggagaaa ttcttcaaat tcccgttgca catagcccgg caattccttt ccctgctctg
      181 ccataagcgc agcgaatgcc gggtaatact cgtcaacgat ctgatagaga agggtttgct
      241 cgggtcggtg gctctggtaa cgaccagtat cccgatcccg gctggccgtc ctggccgcca
      301 catgaggcat gttccgcgtc cttgcaatac tgtgtttaca tacagtctat cgcttagcgg
      361 aaagttcttt taccctcagc cgaaatgcct gccgttgcta gacattgcca gccagtgccc
      421 gtcactcccg tactaactgt cacgaacccc tgcaataact gtcacgcccc cctgcaataa
      481 ctgtcacgaa cccctgcaat aactgtcacg cccccaaacc tgcaaaccca gcaggggcgg
      541 gggctggcgg ggtgttggaa aaatccatcc atgattatct aagaataatc cactaggcgc
      601 ggttatcagc gcccttgtgg ggcgctgctg cccttgccca atatgcccgg ccagaggccg
      661 gatagctggt ctattcgctg cgctaggcta cacaccgccc caccgctgcg cggcaggggg
      721 aaaggcgggc aaagcccgct aaaccccaca ccaaaccccg cagaaatacg ctggagcgct
      781 tttagccgct ttagcggcct ttccccctac ccgaagggtg ggggcgcgtg tgcagccccg
      841 cagggcctgt ctcggtcgat cattcagccc ggctcatcct tctggcgtgg cggcagaccg
      901 aacaaggcgc ggtcgtggtc gcgttcaagg tacgcatcca ttgccgccat gagccgatcc
      961 tccggccact cgctgctgtt caccttggcc aaaatcatgg cccccaccag caccttgcgc
     1021 cttgtttcgt tcttgcgctc ttgctgctgt tcccttgccc gcacccgctg aatttcggca
     1081 ttgattcgcg ctcgttgttc ttcgagcttg gccagccgat ccgccgcctt gttgctcccc
     1141 ttaaccatct tgacacccca ttgttaatgt gctgtctcgt aggctatcat ggagccgaaa
     1201 agcaaaagca acagcgaggc agcatggcga tttatcacct tacggcgaaa accggcagca
     1261 ggtcgggcgg ccaatcggcc agggccaagg ccgactacat ccagcgcgaa ggcaagtatg
     1321 cccgcgacat ggatgaagtc ttgcacgccg aatccgggca catgccggag ttcgtcgagc
     1381 ggcccgccga ctactgggat gctgccgacc tgtatgaacg cgccaatggg cggctgttca
     1441 aggaggtcga atttgccctg ccggtcgagc tgaccctcga ccagcagaag gcgctggcgt
     1501 ccgagttcgc ccagcacctg accggtgccg agcgcctgcc gtatacgctg gccatccatg
     1561 ccggtggcgg cgagaacccg cactgccacc tgatgatctc cgagcggatc aatgacggca
     1621 tcgagcggcc cgccgctcag tggttcaagc ggtacaacgg caagaccccg gagaagggcg
     1681 gggcacagaa gaccgaagcg ctcaagccca aggcatggct tgagcagacc cgcgaggcat
     1741 gggccgacca tgccaaccgg gcattagagc gggctggcca cgacgcccgc attgaccaca
     1801 gaacacttga ggcgcagggc atcgagcgcc tgcccggtgt tcacctgggg ccgaacgtgg
     1861 tggagatgga aggccggggc atccgcaccg accgggcaga cgtggccctg aacatcgaca
     1921 ccgccaacgc ccagatcatc gacttacagg aataccggga ggcaatagac catgaacgca
     1981 atcgacagag tgaagaaatc cagaggcatc aacgagttag cggagcagat cgaaccgctg
     2041 gcccagagca tggcgacact ggccgacgaa gcccggcagg tcatgagcca gacccagcag
     2101 gccagcgagg cgcaggcggc ggagtggctg aaagcccagc gccagacagg ggcggcatgg
     2161 gtggagctgg ccaaagagtt gcgggaggta gccgccgagg tgagcagcgc cgcgcagagc
     2221 gcccggagcg cgtcgcgggg gtggcactgg aagctatggc taaccgtgat gctggcttcc
     2281 atgatgccta cggtggtgct gctgatcgca tcgttgctct tgctcgacct gacgccactg
     2341 acaaccgagg acggctcgat ctggctgcgc ttggtggccc gatgaagaac gacaggactt
     2401 tgcaggccat aggccgacag ctcaaggcca tgggctgtga gcgcttcgat atcggcgtca
     2461 gggacgccac caccggccag atgatgaacc gggaatggtc agccgccgaa gtgctccaga
     2521 acacgccatg gctcaagcgg atgaatgccc agggcaatga cgtgtatatc aggcccgccg
     2581 agcaggagcg gcatggtctg gtgctggtgg acgacctcag cgagtttgac ctggatgaca
     2641 tgaaagccga gggccgggag cctgccctgg tagtggaaac cagcccgaag aactatcagg
     2701 catgggtcaa ggtggccgac gccgcaggcg gtgaacttcg ggggcagatt gcccggacgc
     2761 tggccagcga gtacgacgcc gacccggcca gcgccgacag ccgccactat ggccgcttgg
     2821 cgggcttcac caaccgcaag gacaagcaca ccacccgcgc cggttatcag ccgtgggtgc
     2881 tgctgcgtga atccaagggc aagaccgcca ccgctggccc ggcgctggtg cagcaggctg
     2941 gccagcagat cgagcaggcc cagcggcagc aggagaaggc ccgcaggctg gccagcctcg
     3001 aactgcccga gcggcagctt agccgccacc ggcgcacggc gctggacgag taccgcagcg
     3061 agatggccgg gctggtcaag cgcttcggtg atgacctcag caagtgcgac tttatcgccg
     3121 cgcagaagct ggccagccgg ggccgcagtg ccgaggaaat cggcaaggcc atggccgagg
     3181 ccagcccagc gctggcagag cgcaagcccg gccacgaagc ggattacatc gagcgcaccg
     3241 tcagcaaggt catgggtctg cccagcgtcc agcttgcgcg ggccgagctg gcacgggcac
     3301 cggcaccccg ccagcgaggc atggacaggg gcgggccaga tttcagcatg tagtgcttgc
     3361 gttggtactc acgcctgtta tactatgagt actcacgcac agaagggggt tttatggaat
     3421 acgaaaaaag cgcttcaggg tcggtctacc tgatcaaaag tgacaagggc tattggttgc
     3481 ccggtggctt tggttatacg tcaaacaagg ccgaggctgg ccgcttttca gtcgctgata
     3541 tggccagcct taaccttgac ggctgcacct tgtccttgtt ccgcgaagac aagcctttcg
     3601 gccccggcaa gtttctcggt gactgatatg aaagaccaaa aggacaagca gaccggcgac
     3661 ctgctggcca gccctgacgc tgtacgccaa gcgcgatatg ccgagcgcat gaaggccaaa
     3721 gggatgcgtc agcgcaagtt ctggctgacc gacgacgaat acgaggcgct gcgcgagtgc
     3781 ctggaagaac tcagagcggc gcagggcggg ggtagtgacc ccgccagcgc ctaaccacca
     3841 actgcctgca aaggaggcaa tcaatggcta cccataagcc tatcaatatt ctggaggcgt
     3901 tcgcagcagc gccgccaccg ctggactacg ttttgcccaa catggtggcc ggtacggtcg
     3961 gggcgctggt gtcgcccggt ggtgccggta aatccatgct ggccctgcaa ctggccgcac
     4021 agattgcagg cgggccggat ctgctggagg tgggcgaact gcccaccggc ccggtgatct
     4081 acctgcccgc cgaagacccg cccaccgcca ttcatcaccg cctgcacgcc cttggggcgc
     4141 acctcagcgc cgaggaacgg caagccgtgg ctgacggcct gctgatccag ccgctgatcg
     4201 gcagcctgcc caacatcatg gccccggagt ggttcgacgg cctcaagcgc gccgccgagg
     4261 gccgccgcct gatggtgctg gacacgctgc gccggttcca catcgaggaa gaaaacgcca
     4321 gcggccccat ggcccaggtc atcggtcgca tggaggccat cgccgccgat accgggtgct
     4381 ctatcgtgtt cctgcaccat gccagcaagg gcgcggccat gatgggcgca ggcgaccagc
     4441 agcaggccag ccggggcagc tcggtactgg tcgataacat ccgctggcag tcctacctgt
     4501 cgagcatgac cagcgccgag gccgaggaat ggggtgtgga cgacgaccag cgccggttct
     4561 tcgtccgctt cggtgtgagc aaggccaact atggcgcacc gttcgctgat cggtggttca
     4621 ggcggcatga cggcggggtg ctcaagcccg ccgtgctgga gaggcagcgc aagagcaagg
     4681 gggtgccccg tggtgaagcc taagaacaag cacagcctca gccacgtccg gcacgacccg
     4741 gcgcactgtc tggcccccgg cctgttccgt gccctcaagc ggggcgagcg caagcgcagc
     4801 aagctggacg tgacgtatga ctacggcgac ggcaagcgga tcgagttcag cggcccggag
     4861 ccgctgggcg ctgatgatct gcgcatcctg caagggctgg tggccatggc tgggcctaat
     4921 ggcctagtgc ttggcccgga acccaagacc gaaggcggac ggcagctccg gctgttcctg
     4981 gaacccaagt gggaggccgt caccgctgat gccatggtgg tcaaaggtag ctatcgggcg
     5041 ctggcaaagg aaatcggggc agaggtcgat agtggtgggg cgctcaagca catacaggac
     5101 tgcatcgagc gcctttggaa ggtatccatc atcgcccaga atggccgcaa gcggcagggg
     5161 tttcggctgc tgtcggagta cgccagcgac gaggcggacg ggcgcctgta cgtggccctg
     5221 aaccccttga tcgcgcaggc cgtcatgggt ggcggccagc atgtgcgcat cagcatggac
     5281 gaggtgcggg cgctggacag cgaaaccgcc cgcctgctgc accagcggct gtgtggctgg
     5341 atcgaccccg gcaaaaccgg caaggcttcc atagatacct tgtgcggcta tgtctggccg
     5401 tcagaggcca gtggttcgac catgcgcaag cgccgccagc gggtgcgcga ggcgttgccg
     5461 gagctggtcg cgctgggctg gacggtaacc gagttcgcgg cgggcaagta cgacatcacc
     5521 cggcccaagg cggcaggctg acccccccac tctattgtaa acaagacatt tttatctttt
     5581 atattcaatg gcttattttc ctgctaattg gtaataccat gaaaaatacc atgctcagaa
     5641 aaggcttaac aatattttga aaaattgcct actgagcgct gccgcacagc tccataggcc
     5701 gctttcctgg ctttgcttcc agatgtatgc tcttctgctc ccgaacgcca gcaagacgta
     5761 gcccagcgcg tcggccagct tgcaattcgc gctaacttac attaattgcg ttgcgctcac
     5821 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5881 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     5941 ggcaacagct gattgccctt cacctccaag ctgtccctcc tgttcagcta ctgacggggt
     6001 ggtgcgtaac ggcaaaagca ccgccggaca tcagcgctag cggagtgtat actggcttac
     6061 tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg tggcaggaga aaaaaggctg
     6121 caccggtgcg tcagcagaat atgtgataca ggatatattc cgcttcctcg ctcactgact
     6181 cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt
     6241 tcctggaaga tgccaggaag atacttaaca gggaagtgag agggccgcgg caaagccgtt
     6301 tttccatagg ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg
     6361 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggcggct ccctcgtgcg
     6421 ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc
     6481 tcattccacg cctgacactc agttccgggt aggcagttcg ctccaagctg gactgtatgc
     6541 acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6601 acccggaaag acatgcaaaa gcaccactgg cagcagccac tggtaattga tttagaggag
     6661 ttagtcttga agtcatgcgc cggttaaggc taaactgaaa ggacaagttt tggtgactgc
     6721 gctcctccaa gccagttacc tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa
     6781 ccgccctgca aggcggtttt ttcgttttca gagcaagaga ttacgcgcag accaaaacga
     6841 tctcaagaag atcatcttat taatcagata aaatatttct agagtagtcg actgagaccg
     6901 taaaacgacg gccagtgaat tcttgacggc tagctcagtc ctaggtacag tgctagcttg
     6961 gtctcgatta aagaggagaa agagtcccat ggcttccctg tcaaaacaag ttttgccaag
     7021 agatgtcaga atgcgattcc acatggacgg ttgcgttaat ggtcatcaat ttaccattga
     7081 aggtgaaggt gcaggcaaac cttacgaggg taaaaaaacc ctgaaactca gagtcacaaa
     7141 ggggggtcct ctcccttttg cctttgatat actgagtgct accttcacct atggcaaccg
     7201 atgtttttgc gattaccccg aagatatgcc agattacttt aaacaaagcc tgcccgaggg
     7261 ctattcttgg gagcgtacat taatgttcga ggatggcggc tgcgggaccg cctctgctca
     7321 cataagtctg gaaaaagatt gttttatcca caattccacc tttcacggag tcaacttccc
     7381 agctaatgga cctgtaatgc agaagaagac acttaactgg gaacccagtt cagaacttat
     7441 cactgcttgt gatgggatac tgaaggggga cgttaccatg ttcttattat tggaaggagg
     7501 acatagactt aaatgccaat ttacaacatc atacaaggcc cacaaggccg taaagatgcc
     7561 ccccaaccac attattgaac acgtcctggt gaagaaagaa gtagctgatg gatttcaaat
     7621 ccaggaacac gcagttgcta aacacttcac agtcgatgtc aaggaaacac atcaccacca
     7681 tcaccattga taagagctca aaaaaaaacc ccgcccctga cagggcgggg ttttttttgg
     7741 taccgtcata gctgtttcct gctccccaac ggcgtagagg atcgagatct tttgggttgt
     7801 cactcatcgt atttggttta taattttaag ctaataacct aattataact aattaatagt
     7861 tttgtatctt gaaaaaatag ctataaaact tatttaaata acgaagattt ttgtgtataa
     7921 aatatttata acaaaaaaag gagactaaac atatgagcca tattcaacgg gaaacgtctt
     7981 gctcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc
     8041 gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc
     8101 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg
     8161 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta
     8221 ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat
     8281 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc
     8341 ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg
     8401 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc
     8461 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac
     8521 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga
     8581 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg
     8641 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa
     8701 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt
     8761 ttttctaagg atccggctgc taacaaagc
//
