LOCUS       sequence-472134-        8627 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 77 to 111)
  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 8627)
  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: pKEK3394
FEATURES             Location/Qualifiers
     source          1..8627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     54..70
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        77..111
                     /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             124..135
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(884..900)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(884..900)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1092..1769
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     ADPA"
     primer_bind     complement(1158..1177)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2223..2617
                     /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             4162..5133
                     /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             5644..6483
                     /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             6470..7321
                     /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(7948..7965)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8082..8627)
                     /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(8162..8181)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcatc ttattaatca gataaaatat ttctagagta gtcgactgag accgtaaaac
       61 gacggccagt gaattcttga cggctagctc agtcctaggt acagtgctag cttggtctcg
      121 attaaagagg agaaagagtc ccatggcgag cttagtcaag aaagacatgt gtgtaaagat
      181 gacgatggag gggactgtta atggttacca ctttaagtgt gtgggggaag gtgaggggaa
      241 accgtttgag ggaactcaaa acatgcgtat tcgcgtcacg gagggtggac ctttaccgtt
      301 cgctttcgat attctggcac catgttgcat gtatgggagc aaaaccttta ttaaacatgt
      361 cagcggcatc ccagactatt ttaaagagtc gttccccgaa gggttcacct gggagcgtac
      421 acagatcttc gaggatggcg gagtattgac cgcacatcag gatacaagtc ttgagggcaa
      481 ttgcttgatt tacaaagtga aagtgcttgg gacgaatttc cctgccaacg gacccgttat
      541 gcaaaagaaa actgcaggct gggagccgtg tgttgagatg ctttacccac gtgacggggt
      601 attgtgcggc caaagtctga tggcactgaa atgtaccgat ggtaatcact taacgtcgca
      661 cttacgtaca acataccgta gccgtaagcc ctccaacgcg gtcaacatgc ctgagtttca
      721 ctttggcgac caccgcattg agatcttaaa ggcggaacag gggaaatttt atgaacagta
      781 tgagtcggct gtggcccgtt actctgatgt gcctgagaaa gcaacttaat gagagctcaa
      841 aaaaaaaccc cgcccctgac agggcggggt tttttttggt accgtcatag ctgtttcctg
      901 ctccccaacg gcgtagagga tcgagatctt ttgggttgtc actcatcgta tttggtttat
      961 aattttaagc taataaccta attataacta attaatagtt ttgtatcttg aaaaaatagc
     1021 tataaaactt atttaaataa cgaagatttt tgtgtataaa atatttataa caaaaaaagg
     1081 agactaaaca tatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc
     1141 atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg
     1201 ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc
     1261 cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa
     1321 tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg
     1381 agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc
     1441 tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag
     1501 ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg
     1561 atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt
     1621 atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg
     1681 atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg
     1741 gggggcgtaa acgccgcgcg gatccggctg ctaacaaagc gcgagcctgt cagaccaagt
     1801 ttactcatat atactttaga ttgatttctg aaagcgacca ggtgctcggc gtggcaagac
     1861 tcgcagcgaa cccgtagaaa gccatgctcc agccgcccgc attggagaaa ttcttcaaat
     1921 tcccgttgca catagcccgg caattccttt ccctgctctg ccataagcgc agcgaatgcc
     1981 gggtaatact cgtcaacgat ctgatagaga agggtttgct cgggtcggtg gctctggtaa
     2041 cgaccagtat cccgatcccg gctggccgtc ctggccgcca catgaggcat gttccgcgtc
     2101 cttgcaatac tgtgtttaca tacagtctat cgcttagcgg aaagttcttt taccctcagc
     2161 cgaaatgcct gccgttgcta gacattgcca gccagtgccc gtcactcccg tactaactgt
     2221 cacgaacccc tgcaataact gtcacgcccc cctgcaataa ctgtcacgaa cccctgcaat
     2281 aactgtcacg cccccaaacc tgcaaaccca gcaggggcgg gggctggcgg ggtgttggaa
     2341 aaatccatcc atgattatct aagaataatc cactaggcgc ggttatcagc gcccttgtgg
     2401 ggcgctgctg cccttgccca atatgcccgg ccagaggccg gatagctggt ctattcgctg
     2461 cgctaggcta cacaccgccc caccgctgcg cggcaggggg aaaggcgggc aaagcccgct
     2521 aaaccccaca ccaaaccccg cagaaatacg ctggagcgct tttagccgct ttagcggcct
     2581 ttccccctac ccgaagggtg ggggcgcgtg tgcagccccg cagggcctgt ctcggtcgat
     2641 cattcagccc ggctcatcct tctggcgtgg cggcagaccg aacaaggcgc ggtcgtggtc
     2701 gcgttcaagg tacgcatcca ttgccgccat gagccgatcc tccggccact cgctgctgtt
     2761 caccttggcc aaaatcatgg cccccaccag caccttgcgc cttgtttcgt tcttgcgctc
     2821 ttgctgctgt tcccttgccc gcacccgctg aatttcggca ttgattcgcg ctcgttgttc
     2881 ttcgagcttg gccagccgat ccgccgcctt gttgctcccc ttaaccatct tgacacccca
     2941 ttgttaatgt gctgtctcgt aggctatcat ggagccgaaa agcaaaagca acagcgaggc
     3001 agcatggcga tttatcacct tacggcgaaa accggcagca ggtcgggcgg ccaatcggcc
     3061 agggccaagg ccgactacat ccagcgcgaa ggcaagtatg cccgcgacat ggatgaagtc
     3121 ttgcacgccg aatccgggca catgccggag ttcgtcgagc ggcccgccga ctactgggat
     3181 gctgccgacc tgtatgaacg cgccaatggg cggctgttca aggaggtcga atttgccctg
     3241 ccggtcgagc tgaccctcga ccagcagaag gcgctggcgt ccgagttcgc ccagcacctg
     3301 accggtgccg agcgcctgcc gtatacgctg gccatccatg ccggtggcgg cgagaacccg
     3361 cactgccacc tgatgatctc cgagcggatc aatgacggca tcgagcggcc cgccgctcag
     3421 tggttcaagc ggtacaacgg caagaccccg gagaagggcg gggcacagaa gaccgaagcg
     3481 ctcaagccca aggcatggct tgagcagacc cgcgaggcat gggccgacca tgccaaccgg
     3541 gcattagagc gggctggcca cgacgcccgc attgaccaca gaacacttga ggcgcagggc
     3601 atcgagcgcc tgcccggtgt tcacctgggg ccgaacgtgg tggagatgga aggccggggc
     3661 atccgcaccg accgggcaga cgtggccctg aacatcgaca ccgccaacgc ccagatcatc
     3721 gacttacagg aataccggga ggcaatagac catgaacgca atcgacagag tgaagaaatc
     3781 cagaggcatc aacgagttag cggagcagat cgaaccgctg gcccagagca tggcgacact
     3841 ggccgacgaa gcccggcagg tcatgagcca gacccagcag gccagcgagg cgcaggcggc
     3901 ggagtggctg aaagcccagc gccagacagg ggcggcatgg gtggagctgg ccaaagagtt
     3961 gcgggaggta gccgccgagg tgagcagcgc cgcgcagagc gcccggagcg cgtcgcgggg
     4021 gtggcactgg aagctatggc taaccgtgat gctggcttcc atgatgccta cggtggtgct
     4081 gctgatcgca tcgttgctct tgctcgacct gacgccactg acaaccgagg acggctcgat
     4141 ctggctgcgc ttggtggccc gatgaagaac gacaggactt tgcaggccat aggccgacag
     4201 ctcaaggcca tgggctgtga gcgcttcgat atcggcgtca gggacgccac caccggccag
     4261 atgatgaacc gggaatggtc agccgccgaa gtgctccaga acacgccatg gctcaagcgg
     4321 atgaatgccc agggcaatga cgtgtatatc aggcccgccg agcaggagcg gcatggtctg
     4381 gtgctggtgg acgacctcag cgagtttgac ctggatgaca tgaaagccga gggccgggag
     4441 cctgccctgg tagtggaaac cagcccgaag aactatcagg catgggtcaa ggtggccgac
     4501 gccgcaggcg gtgaacttcg ggggcagatt gcccggacgc tggccagcga gtacgacgcc
     4561 gacccggcca gcgccgacag ccgccactat ggccgcttgg cgggcttcac caaccgcaag
     4621 gacaagcaca ccacccgcgc cggttatcag ccgtgggtgc tgctgcgtga atccaagggc
     4681 aagaccgcca ccgctggccc ggcgctggtg cagcaggctg gccagcagat cgagcaggcc
     4741 cagcggcagc aggagaaggc ccgcaggctg gccagcctcg aactgcccga gcggcagctt
     4801 agccgccacc ggcgcacggc gctggacgag taccgcagcg agatggccgg gctggtcaag
     4861 cgcttcggtg atgacctcag caagtgcgac tttatcgccg cgcagaagct ggccagccgg
     4921 ggccgcagtg ccgaggaaat cggcaaggcc atggccgagg ccagcccagc gctggcagag
     4981 cgcaagcccg gccacgaagc ggattacatc gagcgcaccg tcagcaaggt catgggtctg
     5041 cccagcgtcc agcttgcgcg ggccgagctg gcacgggcac cggcaccccg ccagcgaggc
     5101 atggacaggg gcgggccaga tttcagcatg tagtgcttgc gttggtactc acgcctgtta
     5161 tactatgagt actcacgcac agaagggggt tttatggaat acgaaaaaag cgcttcaggg
     5221 tcggtctacc tgatcaaaag tgacaagggc tattggttgc ccggtggctt tggttatacg
     5281 tcaaacaagg ccgaggctgg ccgcttttca gtcgctgata tggccagcct taaccttgac
     5341 ggctgcacct tgtccttgtt ccgcgaagac aagcctttcg gccccggcaa gtttctcggt
     5401 gactgatatg aaagaccaaa aggacaagca gaccggcgac ctgctggcca gccctgacgc
     5461 tgtacgccaa gcgcgatatg ccgagcgcat gaaggccaaa gggatgcgtc agcgcaagtt
     5521 ctggctgacc gacgacgaat acgaggcgct gcgcgagtgc ctggaagaac tcagagcggc
     5581 gcagggcggg ggtagtgacc ccgccagcgc ctaaccacca actgcctgca aaggaggcaa
     5641 tcaatggcta cccataagcc tatcaatatt ctggaggcgt tcgcagcagc gccgccaccg
     5701 ctggactacg ttttgcccaa catggtggcc ggtacggtcg gggcgctggt gtcgcccggt
     5761 ggtgccggta aatccatgct ggccctgcaa ctggccgcac agattgcagg cgggccggat
     5821 ctgctggagg tgggcgaact gcccaccggc ccggtgatct acctgcccgc cgaagacccg
     5881 cccaccgcca ttcatcaccg cctgcacgcc cttggggcgc acctcagcgc cgaggaacgg
     5941 caagccgtgg ctgacggcct gctgatccag ccgctgatcg gcagcctgcc caacatcatg
     6001 gccccggagt ggttcgacgg cctcaagcgc gccgccgagg gccgccgcct gatggtgctg
     6061 gacacgctgc gccggttcca catcgaggaa gaaaacgcca gcggccccat ggcccaggtc
     6121 atcggtcgca tggaggccat cgccgccgat accgggtgct ctatcgtgtt cctgcaccat
     6181 gccagcaagg gcgcggccat gatgggcgca ggcgaccagc agcaggccag ccggggcagc
     6241 tcggtactgg tcgataacat ccgctggcag tcctacctgt cgagcatgac cagcgccgag
     6301 gccgaggaat ggggtgtgga cgacgaccag cgccggttct tcgtccgctt cggtgtgagc
     6361 aaggccaact atggcgcacc gttcgctgat cggtggttca ggcggcatga cggcggggtg
     6421 ctcaagcccg ccgtgctgga gaggcagcgc aagagcaagg gggtgccccg tggtgaagcc
     6481 taagaacaag cacagcctca gccacgtccg gcacgacccg gcgcactgtc tggcccccgg
     6541 cctgttccgt gccctcaagc ggggcgagcg caagcgcagc aagctggacg tgacgtatga
     6601 ctacggcgac ggcaagcgga tcgagttcag cggcccggag ccgctgggcg ctgatgatct
     6661 gcgcatcctg caagggctgg tggccatggc tgggcctaat ggcctagtgc ttggcccgga
     6721 acccaagacc gaaggcggac ggcagctccg gctgttcctg gaacccaagt gggaggccgt
     6781 caccgctgat gccatggtgg tcaaaggtag ctatcgggcg ctggcaaagg aaatcggggc
     6841 agaggtcgat agtggtgggg cgctcaagca catacaggac tgcatcgagc gcctttggaa
     6901 ggtatccatc atcgcccaga atggccgcaa gcggcagggg tttcggctgc tgtcggagta
     6961 cgccagcgac gaggcggacg ggcgcctgta cgtggccctg aaccccttga tcgcgcaggc
     7021 cgtcatgggt ggcggccagc atgtgcgcat cagcatggac gaggtgcggg cgctggacag
     7081 cgaaaccgcc cgcctgctgc accagcggct gtgtggctgg atcgaccccg gcaaaaccgg
     7141 caaggcttcc atagatacct tgtgcggcta tgtctggccg tcagaggcca gtggttcgac
     7201 catgcgcaag cgccgccagc gggtgcgcga ggcgttgccg gagctggtcg cgctgggctg
     7261 gacggtaacc gagttcgcgg cgggcaagta cgacatcacc cggcccaagg cggcaggctg
     7321 acccccccca ctctattgta aacaagacat ttttatcttt tatattcaat ggcttatttt
     7381 cctgctaatt ggtaatacca tgaaaaatac catgctcaga aaaggcttaa caatattttg
     7441 aaaaattgcc tactgagcgc tgccgcacag ctccataggc cgctttcctg gctttgcttc
     7501 cagatgtatg ctcttctgct cccgaacgcc agcaagacgt agcccagcgc gtcggccagc
     7561 ttgcaattcg cgctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     7621 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     7681 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     7741 tcacctccaa gctgtccctc ctgttcagct actgacgggg tggtgcgtaa cggcaaaagc
     7801 accgccggac atcagcgcta gcggagtgta tactggctta ctatgttggc actgatgagg
     7861 gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct gcaccggtgc gtcagcagaa
     7921 tatgtgatac aggatatatt ccgcttcctc gctcactgac tcgctacgct cggtcgttcg
     7981 actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag atgccaggaa
     8041 gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag gctccgcccc
     8101 cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc gacaggacta
     8161 taaagatacc aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt tcctgccttt
     8221 cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac gcctgacact
     8281 cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc ccgttcagtc
     8341 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa
     8401 agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg aagtcatgcg
     8461 ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca agccagttac
     8521 ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc aaggcggttt
     8581 tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaa
//
