LOCUS       sequence-468002-        6326 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 3733 to 3767)
  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 6326)
  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: pKEK3220
FEATURES             Location/Qualifiers
     source          1..6326
                     /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(2042..2719)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     ADPA"
     primer_bind     2634..2653
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     2911..2927
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2911..2927
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(2985..3008)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(3009..3056)
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     RBS             3709..3720
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(3733..3767)
                     /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(3774..3790)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      3844..4389
                     /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     4290..4309
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4506..4523
                     /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 cgcgcggcgt ttacgccccc cctgccactc atcgcagtac tgttgtaatt
     2101 cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca
     2161 gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga
     2221 agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg
     2281 ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt
     2341 aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac
     2401 tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac
     2461 tatcccatat caccagctca ccgtctttca ttgccatacg gaattccgga tgagcattca
     2521 tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg
     2581 tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg
     2641 actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc
     2701 cagtgatttt tttctccata tgtttagtct cctttttttg ttataaatat tttatacaca
     2761 aaaatcttcg ttatttaaat aagttttata gctatttttt caagatacaa aactattaat
     2821 tagttataat taggttatta gcttaaaatt ataaaccaaa tacgatgagt gacaacccaa
     2881 aagatctcga tcctctacgc cgttggggag caggaaacag ctatgacggt accaaaaaaa
     2941 accccgccct gtcaggggcg gggttttttt ttgagctctt atcacttgtc gtcgtcgtcc
     3001 ttgtagtcgg taatgcccgc cgcgttcaca tattcatgca ggaccatgtg atcgcgtttt
     3061 tcgttcggat ctttcgacag cacggtctgg gtgctcagat agtggttatc cggcagcaga
     3121 accgggccat cgccaatcgg ggtgttctgc tgatagtgat ccgccagctg cactgagcca
     3181 tcttccacgt tatggcgcac ggtaaagttc gctttaatgc catttttctg tttatccgcc
     3241 gtaatgtaca cgttatggct gttaaagttg tattccagtt tgtggcccag aatgttacca
     3301 tcttctttaa aatccgtgcc tttcagttca atgcgattca ccagggtatc gccttcgaat
     3361 ttcacaacgg cacgggtttt gtatttgcca tcatctttga aactaatggt gcgttcctgc
     3421 acatagcctt ccggcatcgc gcttttaaaa aaatcatggc gtttcatgtg atccgggtag
     3481 cggctaaaac actgcacgcc gtaggtcagg gtggtcacca gggtcggcca cggcaccggc
     3541 agtttaccgg tggtgcagat aaatttcagg gtcagtttgc caatggtcgc atcgccttca
     3601 ccttcgccac gcacgctaaa tttatggcca ttcacatcgc catccagttc caccagaatc
     3661 ggcaccacgc cggtaaacag ttcttcgcct ttgctgccca tgggactctt tctcctcttt
     3721 aatcgagacc aagctagcac tgtacctagg actgagctag ccgtcaagaa ttcactggcc
     3781 gtcgttttac ggtctcagtc gactactcta gaaatatttt atctgattaa taagatgatc
     3841 ttcttgagat cgttttggtc tgcgcgtaat ctcttgctct gaaaacgaaa aaaccgcctt
     3901 gcagggcggt ttttcgaagg ttctctgagc taccaactct ttgaaccgag gtaactggct
     3961 tggaggagcg cagtcaccaa aacttgtcct ttcagtttag ccttaaccgg cgcatgactt
     4021 caagactaac tcctctaaat caattaccag tggctgctgc cagtggtgct tttgcatgtc
     4081 tttccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg gactgaacgg
     4141 ggggttcgtg catacagtcc agcttggagc gaactgccta cccggaactg agtgtcaggc
     4201 gtggaatgag acaaacgcgg ccataacagc ggaatgacac cggtaaaccg aaaggcagga
     4261 acaggagagc gcacgaggga gccgccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4321 gggtttcgcc accactgatt tgagcgtcag atttcgtgat gcttgtcagg ggggcggagc
     4381 ctatggaaaa acggctttgc cgcggccctc tcacttccct gttaagtatc ttcctggcat
     4441 cttccaggaa atctccgccc cgttcgtaag ccatttccgc tcgccgcagt cgaacgaccg
     4501 agcgtagcga gtcagtgagc gaggaagcgg aatatatcct gtatcacata ttctgctgac
     4561 gcaccggtgc agcctttttt ctcctgccac atgaagcact tcactgacac cctcatcagt
     4621 gccaacatag taagccagta tacactccgc tagcgctgat gtccggcggt gcttttgccg
     4681 ttacgcacca ccccgtcagt agctgaacag gagggacagc taaagctaga aatgtaccaa
     4741 attttaccta ttttaggata cttttcatga agctcttcta tttttcccca gtcctttaat
     4801 agtctacctt tagagtctcg taaatagtta tctttgtgac aggggcctct tttatctttt
     4861 ttaatgtaac tatatgttat tccaacgtca ctattactat tatccaaatc ttttttagca
     4921 tgccagtaag aactttcata acttaactct atctttcgac ctctttgata tacaacaata
     4981 aagctatagc cagtagtaac aacctgtttt acttttgtta aatctattaa cttcttattt
     5041 atttttttat gtttttttga aaatttaaat atttctatat tcattcctac acttcctcaa
     5101 atccaaatgg tagcttatga ttctcttctg gtttcttttc taattttttt atatttgcaa
     5161 taaaaactct ttttctatct ttgatttttt tattgtccca attcctccaa gtatcatcac
     5221 aaaccctttc aatatcatgt aaatgatgat gtctaaatat tgatctgaca taatacagat
     5281 ctaggtctag ttcatcactt aacacaactt ctctaagtct ttcagatgct tcgattggta
     5341 tgtaatcctc tttattttta gtatctaaag gcttttgctt aaattcttct tctgtctctg
     5401 ctaccttact aactgtaaac ttgatatttg taatcttacg accatgtttt ctgtgatgat
     5461 ccttgtcatc ataggttaca aaaatatccg ataattgatt aatctcttct agtgctggta
     5521 ataggaactt atttttaaaa tttgaatatc tgttgctgta acttttaggt aaatcaaaat
     5581 cattaatcat atcatcgaca tacaatacgc aatcaactat attagcatac cctgcttgtt
     5641 cgcctaattt gcttttgaga agtaagtata atctgcttga atacttactt ttaaatgaaa
     5701 atagtaactg tctttctgct ttagtaaagt actcttgtag ttgtatcatg tgtggcatta
     5761 atgaccaatg aaactcgcaa attaaagcac tgcttttagg gtctgcttca atatatgcaa
     5821 accagttagc tatcttcgtt tgttctttat tcagccatac tggcttagac attattgagt
     5881 gcattaattg cttcaatctc actctgttat gcttaacccc tgtagctttt tcaagatcag
     5941 ataggcttat cttatacctg tgaaactctt tatcttctct tttaaccatt gaggcaacta
     6001 agaatattaa gttttgttct tcttttgtaa ggctatactt tcctgcaaca agagtattag
     6061 acatagctat ttctttgcca gcatttacat ttttaacttc tttcatagaa ctagagtcat
     6121 tatctcgata tacaaattct ataaaacttc tattagtaaa acaactactt cataaaaaaa
     6181 agtagtttta acgatacaaa aagtagtttt aaattcaaaa agtgatacaa aaagtagttt
     6241 taaattcaaa aagtgataca aaaagtagtt ttaaattttt taaaaaagtg cttcaaagcc
     6301 ttatgtagca atacttacag aggatt
//
