LOCUS       sequence-472225-        8626 bp    DNA     circular SYN 12-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 8583 to 8617)
  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 8626)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 12, 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..8626
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             4..15
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(764..780)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(764..780)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             972..1649
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     ADPA"
     primer_bind     complement(1038..1057)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2103..2497
                     /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             4042..5013
                     /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             5524..6363
                     /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             6350..7201
                     /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(7827..7844)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7961..8506)
                     /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(8041..8060)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8560..8576
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8583..8617
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
ORIGIN
        1 attaaagagg agaaagagtc ccatggcgag cttagtcaag aaagacatgt gtgtaaagat
       61 gacgatggag gggactgtta atggttacca ctttaagtgt gtgggggaag gtgaggggaa
      121 accgtttgag ggaactcaaa acatgcgtat tcgcgtcacg gagggtggac ctttaccgtt
      181 cgctttcgat attctggcac catgttgcat gtatgggagc aaaaccttta ttaaacatgt
      241 cagcggcatc ccagactatt ttaaagagtc gttccccgaa gggttcacct gggagcgtac
      301 acagatcttc gaggatggcg gagtattgac cgcacatcag gatacaagtc ttgagggcaa
      361 ttgcttgatt tacaaagtga aagtgcttgg gacgaatttc cctgccaacg gacccgttat
      421 gcaaaagaaa actgcaggct gggagccgtg tgttgagatg ctttacccac gtgacggggt
      481 attgtgcggc caaagtctga tggcactgaa atgtaccgat ggtaatcact taacgtcgca
      541 cttacgtaca acataccgta gccgtaagcc ctccaacgcg gtcaacatgc ctgagtttca
      601 ctttggcgac caccgcattg agatcttaaa ggcggaacag gggaaatttt atgaacagta
      661 tgagtcggct gtggcccgtt actctgatgt gcctgagaaa gcaacttaat gagagctcaa
      721 aaaaaaaccc cgcccctgac agggcggggt tttttttggt accgtcatag ctgtttcctg
      781 ctccccaacg gcgtagagga tcgagatctt ttgggttgtc actcatcgta tttggtttat
      841 aattttaagc taataaccta attataacta attaatagtt ttgtatcttg aaaaaatagc
      901 tataaaactt atttaaataa cgaagatttt tgtgtataaa atatttataa caaaaaaagg
      961 agactaaaca tatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc
     1021 atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg
     1081 ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc
     1141 cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa
     1201 tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg
     1261 agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc
     1321 tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag
     1381 ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg
     1441 atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt
     1501 atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg
     1561 atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg
     1621 gggggcgtaa acgccgcgcg gatccggctg ctaacaaagc gcgagcctgt cagaccaagt
     1681 ttactcatat atactttaga ttgatttctg aaagcgacca ggtgctcggc gtggcaagac
     1741 tcgcagcgaa cccgtagaaa gccatgctcc agccgcccgc attggagaaa ttcttcaaat
     1801 tcccgttgca catagcccgg caattccttt ccctgctctg ccataagcgc agcgaatgcc
     1861 gggtaatact cgtcaacgat ctgatagaga agggtttgct cgggtcggtg gctctggtaa
     1921 cgaccagtat cccgatcccg gctggccgtc ctggccgcca catgaggcat gttccgcgtc
     1981 cttgcaatac tgtgtttaca tacagtctat cgcttagcgg aaagttcttt taccctcagc
     2041 cgaaatgcct gccgttgcta gacattgcca gccagtgccc gtcactcccg tactaactgt
     2101 cacgaacccc tgcaataact gtcacgcccc cctgcaataa ctgtcacgaa cccctgcaat
     2161 aactgtcacg cccccaaacc tgcaaaccca gcaggggcgg gggctggcgg ggtgttggaa
     2221 aaatccatcc atgattatct aagaataatc cactaggcgc ggttatcagc gcccttgtgg
     2281 ggcgctgctg cccttgccca atatgcccgg ccagaggccg gatagctggt ctattcgctg
     2341 cgctaggcta cacaccgccc caccgctgcg cggcaggggg aaaggcgggc aaagcccgct
     2401 aaaccccaca ccaaaccccg cagaaatacg ctggagcgct tttagccgct ttagcggcct
     2461 ttccccctac ccgaagggtg ggggcgcgtg tgcagccccg cagggcctgt ctcggtcgat
     2521 cattcagccc ggctcatcct tctggcgtgg cggcagaccg aacaaggcgc ggtcgtggtc
     2581 gcgttcaagg tacgcatcca ttgccgccat gagccgatcc tccggccact cgctgctgtt
     2641 caccttggcc aaaatcatgg cccccaccag caccttgcgc cttgtttcgt tcttgcgctc
     2701 ttgctgctgt tcccttgccc gcacccgctg aatttcggca ttgattcgcg ctcgttgttc
     2761 ttcgagcttg gccagccgat ccgccgcctt gttgctcccc ttaaccatct tgacacccca
     2821 ttgttaatgt gctgtctcgt aggctatcat ggagccgaaa agcaaaagca acagcgaggc
     2881 agcatggcga tttatcacct tacggcgaaa accggcagca ggtcgggcgg ccaatcggcc
     2941 agggccaagg ccgactacat ccagcgcgaa ggcaagtatg cccgcgacat ggatgaagtc
     3001 ttgcacgccg aatccgggca catgccggag ttcgtcgagc ggcccgccga ctactgggat
     3061 gctgccgacc tgtatgaacg cgccaatggg cggctgttca aggaggtcga atttgccctg
     3121 ccggtcgagc tgaccctcga ccagcagaag gcgctggcgt ccgagttcgc ccagcacctg
     3181 accggtgccg agcgcctgcc gtatacgctg gccatccatg ccggtggcgg cgagaacccg
     3241 cactgccacc tgatgatctc cgagcggatc aatgacggca tcgagcggcc cgccgctcag
     3301 tggttcaagc ggtacaacgg caagaccccg gagaagggcg gggcacagaa gaccgaagcg
     3361 ctcaagccca aggcatggct tgagcagacc cgcgaggcat gggccgacca tgccaaccgg
     3421 gcattagagc gggctggcca cgacgcccgc attgaccaca gaacacttga ggcgcagggc
     3481 atcgagcgcc tgcccggtgt tcacctgggg ccgaacgtgg tggagatgga aggccggggc
     3541 atccgcaccg accgggcaga cgtggccctg aacatcgaca ccgccaacgc ccagatcatc
     3601 gacttacagg aataccggga ggcaatagac catgaacgca atcgacagag tgaagaaatc
     3661 cagaggcatc aacgagttag cggagcagat cgaaccgctg gcccagagca tggcgacact
     3721 ggccgacgaa gcccggcagg tcatgagcca gacccagcag gccagcgagg cgcaggcggc
     3781 ggagtggctg aaagcccagc gccagacagg ggcggcatgg gtggagctgg ccaaagagtt
     3841 gcgggaggta gccgccgagg tgagcagcgc cgcgcagagc gcccggagcg cgtcgcgggg
     3901 gtggcactgg aagctatggc taaccgtgat gctggcttcc atgatgccta cggtggtgct
     3961 gctgatcgca tcgttgctct tgctcgacct gacgccactg acaaccgagg acggctcgat
     4021 ctggctgcgc ttggtggccc gatgaagaac gacaggactt tgcaggccat aggccgacag
     4081 ctcaaggcca tgggctgtga gcgcttcgat atcggcgtca gggacgccac caccggccag
     4141 atgatgaacc gggaatggtc agccgccgaa gtgctccaga acacgccatg gctcaagcgg
     4201 atgaatgccc agggcaatga cgtgtatatc aggcccgccg agcaggagcg gcatggtctg
     4261 gtgctggtgg acgacctcag cgagtttgac ctggatgaca tgaaagccga gggccgggag
     4321 cctgccctgg tagtggaaac cagcccgaag aactatcagg catgggtcaa ggtggccgac
     4381 gccgcaggcg gtgaacttcg ggggcagatt gcccggacgc tggccagcga gtacgacgcc
     4441 gacccggcca gcgccgacag ccgccactat ggccgcttgg cgggcttcac caaccgcaag
     4501 gacaagcaca ccacccgcgc cggttatcag ccgtgggtgc tgctgcgtga atccaagggc
     4561 aagaccgcca ccgctggccc ggcgctggtg cagcaggctg gccagcagat cgagcaggcc
     4621 cagcggcagc aggagaaggc ccgcaggctg gccagcctcg aactgcccga gcggcagctt
     4681 agccgccacc ggcgcacggc gctggacgag taccgcagcg agatggccgg gctggtcaag
     4741 cgcttcggtg atgacctcag caagtgcgac tttatcgccg cgcagaagct ggccagccgg
     4801 ggccgcagtg ccgaggaaat cggcaaggcc atggccgagg ccagcccagc gctggcagag
     4861 cgcaagcccg gccacgaagc ggattacatc gagcgcaccg tcagcaaggt catgggtctg
     4921 cccagcgtcc agcttgcgcg ggccgagctg gcacgggcac cggcaccccg ccagcgaggc
     4981 atggacaggg gcgggccaga tttcagcatg tagtgcttgc gttggtactc acgcctgtta
     5041 tactatgagt actcacgcac agaagggggt tttatggaat acgaaaaaag cgcttcaggg
     5101 tcggtctacc tgatcaaaag tgacaagggc tattggttgc ccggtggctt tggttatacg
     5161 tcaaacaagg ccgaggctgg ccgcttttca gtcgctgata tggccagcct taaccttgac
     5221 ggctgcacct tgtccttgtt ccgcgaagac aagcctttcg gccccggcaa gtttctcggt
     5281 gactgatatg aaagaccaaa aggacaagca gaccggcgac ctgctggcca gccctgacgc
     5341 tgtacgccaa gcgcgatatg ccgagcgcat gaaggccaaa gggatgcgtc agcgcaagtt
     5401 ctggctgacc gacgacgaat acgaggcgct gcgcgagtgc ctggaagaac tcagagcggc
     5461 gcagggcggg ggtagtgacc ccgccagcgc ctaaccacca actgcctgca aaggaggcaa
     5521 tcaatggcta cccataagcc tatcaatatt ctggaggcgt tcgcagcagc gccgccaccg
     5581 ctggactacg ttttgcccaa catggtggcc ggtacggtcg gggcgctggt gtcgcccggt
     5641 ggtgccggta aatccatgct ggccctgcaa ctggccgcac agattgcagg cgggccggat
     5701 ctgctggagg tgggcgaact gcccaccggc ccggtgatct acctgcccgc cgaagacccg
     5761 cccaccgcca ttcatcaccg cctgcacgcc cttggggcgc acctcagcgc cgaggaacgg
     5821 caagccgtgg ctgacggcct gctgatccag ccgctgatcg gcagcctgcc caacatcatg
     5881 gccccggagt ggttcgacgg cctcaagcgc gccgccgagg gccgccgcct gatggtgctg
     5941 gacacgctgc gccggttcca catcgaggaa gaaaacgcca gcggccccat ggcccaggtc
     6001 atcggtcgca tggaggccat cgccgccgat accgggtgct ctatcgtgtt cctgcaccat
     6061 gccagcaagg gcgcggccat gatgggcgca ggcgaccagc agcaggccag ccggggcagc
     6121 tcggtactgg tcgataacat ccgctggcag tcctacctgt cgagcatgac cagcgccgag
     6181 gccgaggaat ggggtgtgga cgacgaccag cgccggttct tcgtccgctt cggtgtgagc
     6241 aaggccaact atggcgcacc gttcgctgat cggtggttca ggcggcatga cggcggggtg
     6301 ctcaagcccg ccgtgctgga gaggcagcgc aagagcaagg gggtgccccg tggtgaagcc
     6361 taagaacaag cacagcctca gccacgtccg gcacgacccg gcgcactgtc tggcccccgg
     6421 cctgttccgt gccctcaagc ggggcgagcg caagcgcagc aagctggacg tgacgtatga
     6481 ctacggcgac ggcaagcgga tcgagttcag cggcccggag ccgctgggcg ctgatgatct
     6541 gcgcatcctg caagggctgg tggccatggc tgggcctaat ggcctagtgc ttggcccgga
     6601 acccaagacc gaaggcggac ggcagctccg gctgttcctg gaacccaagt gggaggccgt
     6661 caccgctgat gccatggtgg tcaaaggtag ctatcgggcg ctggcaaagg aaatcggggc
     6721 agaggtcgat agtggtgggg cgctcaagca catacaggac tgcatcgagc gcctttggaa
     6781 ggtatccatc atcgcccaga atggccgcaa gcggcagggg tttcggctgc tgtcggagta
     6841 cgccagcgac gaggcggacg ggcgcctgta cgtggccctg aaccccttga tcgcgcaggc
     6901 cgtcatgggt ggcggccagc atgtgcgcat cagcatggac gaggtgcggg cgctggacag
     6961 cgaaaccgcc cgcctgctgc accagcggct gtgtggctgg atcgaccccg gcaaaaccgg
     7021 caaggcttcc atagatacct tgtgcggcta tgtctggccg tcagaggcca gtggttcgac
     7081 catgcgcaag cgccgccagc gggtgcgcga ggcgttgccg gagctggtcg cgctgggctg
     7141 gacggtaacc gagttcgcgg cgggcaagta cgacatcacc cggcccaagg cggcaggctg
     7201 acccccccac tctattgtaa acaagacatt tttatctttt atattcaatg gcttattttc
     7261 ctgctaattg gtaataccat gaaaaatacc atgctcagaa aaggcttaac aatattttga
     7321 aaaattgcct actgagcgct gccgcacagc tccataggcc gctttcctgg ctttgcttcc
     7381 agatgtatgc tcttctgctc ccgaacgcca gcaagacgta gcccagcgcg tcggccagct
     7441 tgcaattcgc gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     7501 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     7561 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     7621 cacctccaag ctgtccctcc tgttcagcta ctgacggggt ggtgcgtaac ggcaaaagca
     7681 ccgccggaca tcagcgctag cggagtgtat actggcttac tatgttggca ctgatgaggg
     7741 tgtcagtgaa gtgcttcatg tggcaggaga aaaaaggctg caccggtgcg tcagcagaat
     7801 atgtgataca ggatatattc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga
     7861 ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt tcctggaaga tgccaggaag
     7921 atacttaaca gggaagtgag agggccgcgg caaagccgtt tttccatagg ctccgccccc
     7981 ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat
     8041 aaagatacca ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc
     8101 ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc
     8161 agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc
     8221 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa
     8281 gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc
     8341 cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc
     8401 tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt
     8461 ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat
     8521 taatcagata aaatatttct agagtagtcg actgagaccg taaaacgacg gccagtgaat
     8581 tcttgacggc tagctcagtc ctaggtacag tgctagcttg gtctcg
//
