LOCUS       sequence-471771-        8820 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 8710 to 8744)
  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 8820)
  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: pKEK3225
FEATURES             Location/Qualifiers
     source          1..8820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..546
                     /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     447..466
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     663..680
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1307..2158)
                     /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"
     CDS             complement(2145..2984)
                     /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             complement(3495..4466)
                     /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"
     rep_origin      6011..6405
                     /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             complement(6869..7684)
                     /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(6869..7684)
                     /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     7592..7611
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7876..7892
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7876..7892
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(7950..7967)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             8686..8697
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(8710..8744)
                     /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(8751..8767)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa ccgccttgca
       61 gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta actggcttgg
      121 aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taaccggcgc atgacttcaa
      181 gactaactcc tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt gcatgtcttt
      241 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcggac tgaacggggg
      301 gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc ggaactgagt gtcaggcgtg
      361 gaatgagaca aacgcggcca taacagcgga atgacaccgg taaaccgaaa ggcaggaaca
      421 ggagagcgca cgagggagcc gccaggggga aacgcctggt atctttatag tcctgtcggg
      481 tttcgccacc actgatttga gcgtcagatt tcgtgatgct tgtcaggggg gcggagccta
      541 tggaaaaacg gctttgccgc ggccctctca cttccctgtt aagtatcttc ctggcatctt
      601 ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga acgaccgagc
      661 gtagcgagtc agtgagcgag gaagcggaat atatcctgta tcacatattc tgctgacgca
      721 ccggtgcagc cttttttctc ctgccacatg aagcacttca ctgacaccct catcagtgcc
      781 aacatagtaa gccagtatac actccgctag cgctgatgtc cggcggtgct tttgccgtta
      841 cgcaccaccc cgtcagtagc tgaacaggag ggacagcttg gaggtgaagg gcaatcagct
      901 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
      961 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     1021 cgggcagtga gcgcaacgca attaatgtaa gttagcgcga attgcaagct ggccgacgcg
     1081 ctgggctacg tcttgctggc gttcgggagc agaagagcat acatctggaa gcaaagccag
     1141 gaaagcggcc tatggagctg tgcggcagcg ctcagtaggc aatttttcaa aatattgtta
     1201 agccttttct gagcatggta tttttcatgg tattaccaat tagcaggaaa ataagccatt
     1261 gaatataaaa gataaaaatg tcttgtttac aatagagtgg ggggggtcag cctgccgcct
     1321 tgggccgggt gatgtcgtac ttgcccgccg cgaactcggt taccgtccag cccagcgcga
     1381 ccagctccgg caacgcctcg cgcacccgct ggcggcgctt gcgcatggtc gaaccactgg
     1441 cctctgacgg ccagacatag ccgcacaagg tatctatgga agccttgccg gttttgccgg
     1501 ggtcgatcca gccacacagc cgctggtgca gcaggcgggc ggtttcgctg tccagcgccc
     1561 gcacctcgtc catgctgatg cgcacatgct ggccgccacc catgacggcc tgcgcgatca
     1621 aggggttcag ggccacgtac aggcgcccgt ccgcctcgtc gctggcgtac tccgacagca
     1681 gccgaaaccc ctgccgcttg cggccattct gggcgatgat ggataccttc caaaggcgct
     1741 cgatgcagtc ctgtatgtgc ttgagcgccc caccactatc gacctctgcc ccgatttcct
     1801 ttgccagcgc ccgatagcta cctttgacca ccatggcatc agcggtgacg gcctcccact
     1861 tgggttccag gaacagccgg agctgccgtc cgccttcggt cttgggttcc gggccaagca
     1921 ctaggccatt aggcccagcc atggccacca gcccttgcag gatgcgcaga tcatcagcgc
     1981 ccagcggctc cgggccgctg aactcgatcc gcttgccgtc gccgtagtca tacgtcacgt
     2041 ccagcttgct gcgcttgcgc tcgccccgct tgagggcacg gaacaggccg ggggccagac
     2101 agtgcgccgg gtcgtgccgg acgtggctga ggctgtgctt gttcttaggc ttcaccacgg
     2161 ggcaccccct tgctcttgcg ctgcctctcc agcacggcgg gcttgagcac cccgccgtca
     2221 tgccgcctga accaccgatc agcgaacggt gcgccatagt tggccttgct cacaccgaag
     2281 cggacgaaga accggcgctg gtcgtcgtcc acaccccatt cctcggcctc ggcgctggtc
     2341 atgctcgaca ggtaggactg ccagcggatg ttatcgacca gtaccgagct gccccggctg
     2401 gcctgctgct ggtcgcctgc gcccatcatg gccgcgccct tgctggcatg gtgcaggaac
     2461 acgatagagc acccggtatc ggcggcgatg gcctccatgc gaccgatgac ctgggccatg
     2521 gggccgctgg cgttttcttc ctcgatgtgg aaccggcgca gcgtgtccag caccatcagg
     2581 cggcggccct cggcggcgcg cttgaggccg tcgaaccact ccggggccat gatgttgggc
     2641 aggctgccga tcagcggctg gatcagcagg ccgtcagcca cggcttgccg ttcctcggcg
     2701 ctgaggtgcg ccccaagggc gtgcaggcgg tgatgaatgg cggtgggcgg gtcttcggcg
     2761 ggcaggtaga tcaccgggcc ggtgggcagt tcgcccacct ccagcagatc cggcccgcct
     2821 gcaatctgtg cggccagttg cagggccagc atggatttac cggcaccacc gggcgacacc
     2881 agcgccccga ccgtaccggc caccatgttg ggcaaaacgt agtccagcgg tggcggcgct
     2941 gctgcgaacg cctccagaat attgataggc ttatgggtag ccattgattg cctcctttgc
     3001 aggcagttgg tggttaggcg ctggcggggt cactaccccc gccctgcgcc gctctgagtt
     3061 cttccaggca ctcgcgcagc gcctcgtatt cgtcgtcggt cagccagaac ttgcgctgac
     3121 gcatcccttt ggccttcatg cgctcggcat atcgcgcttg gcgtacagcg tcagggctgg
     3181 ccagcaggtc gccggtctgc ttgtcctttt ggtctttcat atcagtcacc gagaaacttg
     3241 ccggggccga aaggcttgtc ttcgcggaac aaggacaagg tgcagccgtc aaggttaagg
     3301 ctggccatat cagcgactga aaagcggcca gcctcggcct tgtttgacgt ataaccaaag
     3361 ccaccgggca accaatagcc cttgtcactt ttgatcaggt agaccgaccc tgaagcgctt
     3421 ttttcgtatt ccataaaacc cccttctgtg cgtgagtact catagtataa caggcgtgag
     3481 taccaacgca agcactacat gctgaaatct ggcccgcccc tgtccatgcc tcgctggcgg
     3541 ggtgccggtg cccgtgccag ctcggcccgc gcaagctgga cgctgggcag acccatgacc
     3601 ttgctgacgg tgcgctcgat gtaatccgct tcgtggccgg gcttgcgctc tgccagcgct
     3661 gggctggcct cggccatggc cttgccgatt tcctcggcac tgcggccccg gctggccagc
     3721 ttctgcgcgg cgataaagtc gcacttgctg aggtcatcac cgaagcgctt gaccagcccg
     3781 gccatctcgc tgcggtactc gtccagcgcc gtgcgccggt ggcggctaag ctgccgctcg
     3841 ggcagttcga ggctggccag cctgcgggcc ttctcctgct gccgctgggc ctgctcgatc
     3901 tgctggccag cctgctgcac cagcgccggg ccagcggtgg cggtcttgcc cttggattca
     3961 cgcagcagca cccacggctg ataaccggcg cgggtggtgt gcttgtcctt gcggttggtg
     4021 aagcccgcca agcggccata gtggcggctg tcggcgctgg ccgggtcggc gtcgtactcg
     4081 ctggccagcg tccgggcaat ctgcccccga agttcaccgc ctgcggcgtc ggccaccttg
     4141 acccatgcct gatagttctt cgggctggtt tccactacca gggcaggctc ccggccctcg
     4201 gctttcatgt catccaggtc aaactcgctg aggtcgtcca ccagcaccag accatgccgc
     4261 tcctgctcgg cgggcctgat atacacgtca ttgccctggg cattcatccg cttgagccat
     4321 ggcgtgttct ggagcacttc ggcggctgac cattcccggt tcatcatctg gccggtggtg
     4381 gcgtccctga cgccgatatc gaagcgctca cagcccatgg ccttgagctg tcggcctatg
     4441 gcctgcaaag tcctgtcgtt cttcatcggg ccaccaagcg cagccagatc gagccgtcct
     4501 cggttgtcag tggcgtcagg tcgagcaaga gcaacgatgc gatcagcagc accaccgtag
     4561 gcatcatgga agccagcatc acggttagcc atagcttcca gtgccacccc cgcgacgcgc
     4621 tccgggcgct ctgcgcggcg ctgctcacct cggcggctac ctcccgcaac tctttggcca
     4681 gctccaccca tgccgcccct gtctggcgct gggctttcag ccactccgcc gcctgcgcct
     4741 cgctggcctg ctgggtctgg ctcatgacct gccgggcttc gtcggccagt gtcgccatgc
     4801 tctgggccag cggttcgatc tgctccgcta actcgttgat gcctctggat ttcttcactc
     4861 tgtcgattgc gttcatggtc tattgcctcc cggtattcct gtaagtcgat gatctgggcg
     4921 ttggcggtgt cgatgttcag ggccacgtct gcccggtcgg tgcggatgcc ccggccttcc
     4981 atctccacca cgttcggccc caggtgaaca ccgggcaggc gctcgatgcc ctgcgcctca
     5041 agtgttctgt ggtcaatgcg ggcgtcgtgg ccagcccgct ctaatgcccg gttggcatgg
     5101 tcggcccatg cctcgcgggt ctgctcaagc catgccttgg gcttgagcgc ttcggtcttc
     5161 tgtgccccgc ccttctccgg ggtcttgccg ttgtaccgct tgaaccactg agcggcgggc
     5221 cgctcgatgc cgtcattgat ccgctcggag atcatcaggt ggcagtgcgg gttctcgccg
     5281 ccaccggcat ggatggccag cgtatacggc aggcgctcgg caccggtcag gtgctgggcg
     5341 aactcggacg ccagcgcctt ctgctggtcg agggtcagct cgaccggcag ggcaaattcg
     5401 acctccttga acagccgccc attggcgcgt tcatacaggt cggcagcatc ccagtagtcg
     5461 gcgggccgct cgacgaactc cggcatgtgc ccggattcgg cgtgcaagac ttcatccatg
     5521 tcgcgggcat acttgccttc gcgctggatg tagtcggcct tggccctggc cgattggccg
     5581 cccgacctgc tgccggtttt cgccgtaagg tgataaatcg ccatgctgcc tcgctgttgc
     5641 ttttgctttt cggctccatg atagcctacg agacagcaca ttaacaatgg ggtgtcaaga
     5701 tggttaaggg gagcaacaag gcggcggatc ggctggccaa gctcgaagaa caacgagcgc
     5761 gaatcaatgc cgaaattcag cgggtgcggg caagggaaca gcagcaagag cgcaagaacg
     5821 aaacaaggcg caaggtgctg gtgggggcca tgattttggc caaggtgaac agcagcgagt
     5881 ggccggagga tcggctcatg gcggcaatgg atgcgtacct tgaacgcgac cacgaccgcg
     5941 ccttgttcgg tctgccgcca cgccagaagg atgagccggg ctgaatgatc gaccgagaca
     6001 ggccctgcgg ggctgcacac gcgcccccac ccttcgggta gggggaaagg ccgctaaagc
     6061 ggctaaaagc gctccagcgt atttctgcgg ggtttggtgt ggggtttagc gggctttgcc
     6121 cgcctttccc cctgccgcgc agcggtgggg cggtgtgtag cctagcgcag cgaatagacc
     6181 agctatccgg cctctggccg ggcatattgg gcaagggcag cagcgcccca caagggcgct
     6241 gataaccgcg cctagtggat tattcttaga taatcatgga tggatttttc caacaccccg
     6301 ccagcccccg cccctgctgg gtttgcaggt ttgggggcgt gacagttatt gcaggggttc
     6361 gtgacagtta ttgcaggggg gcgtgacagt tattgcaggg gttcgtgaca gttagtacgg
     6421 gagtgacggg cactggctgg caatgtctag caacggcagg catttcggct gagggtaaaa
     6481 gaactttccg ctaagcgata gactgtatgt aaacacagta ttgcaaggac gcggaacatg
     6541 cctcatgtgg cggccaggac ggccagccgg gatcgggata ctggtcgtta ccagagccac
     6601 cgacccgagc aaacccttct ctatcagatc gttgacgagt attacccggc attcgctgcg
     6661 cttatggcag agcagggaaa ggaattgccg ggctatgtgc aacgggaatt tgaagaattt
     6721 ctccaatgcg ggcggctgga gcatggcttt ctacgggttc gctgcgagtc ttgccacgcc
     6781 gagcacctgg tcgctttcag aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6841 ggctcgcgct ttgttagcag ccggatcctt agaaaaactc atcgagcatc aaatgaaact
     6901 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     6961 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     7021 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     7081 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agcttatgca
     7141 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     7201 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     7261 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     7321 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     7381 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     7441 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     7501 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     7561 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     7621 cagcatccat gttggaattt aatcgcggcc tcgagcaaga cgtttcccgt tgaatatggc
     7681 tcatatgttt agtctccttt ttttgttata aatattttat acacaaaaat cttcgttatt
     7741 taaataagtt ttatagctat tttttcaaga tacaaaacta ttaattagtt ataattaggt
     7801 tattagctta aaattataaa ccaaatacga tgagtgacaa cccaaaagat ctcgatcctc
     7861 tacgccgttg gggagcagga aacagctatg acggtaccaa aaaaaacccc gccctgtcag
     7921 gggcggggtt tttttttgag ctcttatcag tgatgatgat gatggtgctg atatgtgtct
     7981 aagtctacgg cttttacaac ttctacgagg cacatgtgat ctcttctctc atctctatct
     8041 ttagataatt tagtgtggta agtgatatga tgataccttg gtatatgcac agcagcactt
     8101 ccggcgagag gcctgttcat ctgggaacac ttggtgacca gtttttcagt caccccctca
     8161 atatcgtatg cttgattaaa ttccactcgg attccattgt taacggtatc gggcagtata
     8221 tacaggatgc tgggaggaca ttgaaaagca acgttcttcc tcagaatgtg tccatccttt
     8281 ttgaagtttt ctccggtcaa ggtcaccctg ttgtatatag accctctctc ataggttacc
     8341 atagctcttg tcttatatac gccgtcgcct tcaaaactta tagttctttc ttgggtgtaa
     8401 ccctctggca ttgctgactt aaagaaatct tttatatggc tggggtactt agcaaaacat
     8461 tggaccccat agctaagggt tgacaccagt gtagcccagg gtactggtaa atcgccagtt
     8521 gtacagatat atttggcctt aatagtacca gtggtagcgt cgccagttcc ctcccccttg
     8581 attatgaact tcatgccctc cacatcccct tcaagttctg ttatgtaggg tatttctttt
     8641 tcaaacagtt tagcaccctc tgtcaaggca gtagccatgg gactctttct cctctttaat
     8701 cgagaccaag ctagcactgt acctaggact gagctagccg tcaagaattc actggccgtc
     8761 gttttacggt ctcagtcgac tactctagaa atattttatc tgattaataa gatgatcttc
//
