LOCUS       sequence-337524-        6776 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express colicinB-His6, V21C, M261C.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6776)
  AUTHORS   Skare JT, Roof SK, Postle K
  TITLE     A mutation in the amino terminus of a hybrid TrpC-TonB protein 
            relieves overproduction lethality and results in cytoplasmic 
            accumulation.
  JOURNAL   J Bacteriol. 1989 Aug;171(8):4442-7. doi: 
            10.1128/jb.171.8.4442-4447.1989.
  PUBMED    2546922
REFERENCE   2  (bases 1 to 6776)
  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"; doi: 
            "10.1128/jb.171.8.4442-4447"; journalName: "J Bacteriol"; date: 
            "1989-08"; volume: "171"; issue: "8"; pages: "4442-7"
COMMENT     Sequence Label: pKP933
FEATURES             Location/Qualifiers
     source          1..6776
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..23,24..29)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             2118..2135
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2520..2531
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2960..2979)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4832..5491)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5406..5425
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5492..5594)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5986..6003)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6120..6665)
                     /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(6200..6219)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttgacagctt atcatcgata agctttaatg cggtagttta tcacagttaa attgctaacg
       61 cagtcaggca ccgtgtatga aatctaacaa tgcgctcatc gtcatcctcg gcaccgtcac
      121 cctggatgct gtaggcatag gcttggttat gccggtactg ccgggcctct tgcgggatat
      181 cgtccattcc gacagcatcg ccagtcacta tggcgtgctg ctagcgctat atgcgttgat
      241 gcaatttcta tgcgcacccg ttctcggagc actgtccgac cgctttggcc gccgcccagt
      301 cctgctcgct tcgctacttg gagccactat cgactacgcg atcatggcga ccacacccgt
      361 cctgtggatc cagaacagtt atgcctgtgc cgttttttgt ggtatcaggc gagaatagca
      421 cgtggtgtga atgcctgttt tttgatcgtt ttcacaaaaa ttgaaatcca cagtcttgac
      481 agagaaaaca cagcggcgta atttttgatc tgtacataaa accagtggtt atatgtacag
      541 tatttaattt taattgattg tttttaaagt caaagaggtt ttctatgagt gataatgaag
      601 gtagtgtacc gacagaaggt attgattacg gggacacaat ggtttgttgg ccgtcaacag
      661 gacgaattcc gggcggtgat gtgaaacccg gaggctcatc aggtctcgct ccatccatgc
      721 ctccgggatg gggggattac agcccacaag gtatcgcact tgtacaaagt gttctttttc
      781 ctggaattat tcgccggatt attcttgata aagaacttga agagggagac tggtcgggat
      841 ggtctgtcag tgtgcatagc ccctggggaa acgagaaagt ttccgctgca cgaacagttc
      901 ttgaaaatgg tttacgtggt ggtttgccag aaccgtctcg cccggctgct gtttcttttg
      961 cccgtctgga gcctgcttcc ggaaatgagc aaaaaattat tcgtcttatg gttacacagc
     1021 aactggagca ggtaacggat atccctgcca gccagttacc agcagcgggt aataatgtac
     1081 cggtaaaata tcgtctgacg gaccttatgc agaatggtac acaatatatg gctattatcg
     1141 gaggtattcc gatgacagtg ccagtagtgg atgccgttcc agttccggac cggagtcgtc
     1201 cgggaaccaa tattaaagat gtttacagtg cccctgtatc accaaatcta ccggacctgg
     1261 tattaagtgt gggtcagatg aatactccag ttcggtctaa tcccgaaatc caggaagatg
     1321 gcgttatttc tgagacaggg aattatgttg aggctggtta tacgtgttcc agtaataatc
     1381 atgatgtcat tgtccgtttt cctgaaggca gtggagtttc tccgctatat atttcagccg
     1441 tggagattct ggacagtaat agtttaagcc agcgccagga agccgaaaat aacgcaaagg
     1501 atgacttcag agtcaagaaa gaacaagaaa atgacgagaa gacggtcctg acaaaaacca
     1561 gcgaggtcat cattagtgtc ggtgacaaag tcggggaata tcttggagat aaatacaagg
     1621 cgctttcccg tgaaattgca gagaatataa ataattttca gggaaaaacg attcgtagtt
     1681 atgatgatgc aatgtcttcc attaataagt taatggctaa ccccagcctt aaaataaatg
     1741 caacggacaa agaagccatt gtgaatgcgt ggaaagcatt taatgctgag gatatgggga
     1801 ataaatttgc tgcgttgggt aaaacgttca aagcagcaga ttatgcaata aaggcaaaca
     1861 acatcaggga gaagagtatt gagggttacc agactggtaa ctgggggcca ttaatgctgg
     1921 aagtcgagtc ctgggttatc agtgggatgg catctgctgt agctcttagt ttgttttctt
     1981 tgacattagg ctcggccctt atagcctttg gtctttcggc cacagttgtt ggttttgttg
     2041 gcgtagttat tgcaggtgct attggtgcat ttatcgatga taaatttgtt gatgagttga
     2101 atcacaagat tataaaacac catcaccatc accattaatt aaaaggaagg cagtgttact
     2161 gcctcccttt aaaatattta atgacaagta atggcatcag taaaatcgca tatgtcatgg
     2221 ttaaaacagt aaagtaaatg gagcaatata aaataaacag agtgatgaca ttatgagata
     2281 ataatttaaa aaaaccacct gctgttgtta gttccagatt atagaacagt gtgatataaa
     2341 tcagaatggc ataaaaaacg ggcccccaga aacaggattg cacaacaagt tttcttgtta
     2401 ctggctttat gatttctctt gtcttatatg tgcaaagaaa aacgagaata cccataactg
     2461 gcgctgtctt gttgtagttt gacataagtg ttgtcatgac gggatcatgc aatgatgtaa
     2521 tagaagggag aaatgccacc ttattatata agtagtacag cacttgagaa tatggatcat
     2581 ttattcgaag tattaatata gccattaatg gaataatccc gattatggaa aatgcatata
     2641 atatttcgtt tgctttcttg ttcttatctt tattgctggt catatatata cctttttgta
     2701 ttttatatat agattatata tcagagttct caatatattt ccagtattta atggagtgta
     2761 cagatatata tcttatatat ggtagttcag gagctgccct accattttta acggcatatc
     2821 aggcaaacaa tgttatgggg agcctctctc atggggtttg ttcgccggcg gaggcgttag
     2881 ccagcggcga actctgggat cctctacgcc ggacgcatcg tggccggcat caccggcgcc
     2941 acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc
     3001 cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
     3061 ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc
     3121 ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgaccg
     3181 atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc
     3241 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg
     3301 ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg
     3361 cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc
     3421 aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccgacgc gctgggctac
     3481 gtcttgctgg cgttcgcgac gcgaggctgg atggccttcc ccattatgat tcttctcgct
     3541 tccggcggca tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt agatgacgac
     3601 catcagggac agcttcaagg atcgctcgcg gctcttacca gcctaacttc gatcattgga
     3661 ccgctgatcg tcacggcgat ttatgccgcc tcggcgagca catggaacgg gttggcatgg
     3721 attgtaggcg ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc
     3781 cgggccacct cgacctgaat ggaagccggc ggcacctcgc taacggattc accactccaa
     3841 gaattggagc caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga
     3901 acatatccat cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg ggcagcgttg
     3961 ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc
     4021 ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact
     4081 gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt
     4141 cgtaaagtct ggaaacgcgg aagtccccta cgtgctgctg aagttgcccg caacagagag
     4201 tggaaccaac cggtgatacc acgatactat gactgagagt caacgccatg agcggcctca
     4261 tttcttattc tgagttacaa cagtccgcac cgctgccggt agctccttcc ggtgggcgcg
     4321 gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca
     4381 ggtgccggca gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     4441 acaagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg
     4501 tggaacacct acatctgtat taacgaagcg ctaaccgttt ttatcaggct ctgggaggca
     4561 gaataaatga tcatatcgtc aattattacc tccacgggga gagcctgagc aaactggcct
     4621 caggcatttg agaagcacac ggtcacactg cttccggtag tcaataaacc ggtaaaccag
     4681 caatagacat aagcggctat ttaacgaccc tgccctgaac cgacgaccgg gtcgaatttg
     4741 ctttcgaatt tctgccattc atccgcttat tatcacttat tcaggcgtag caaccaggcg
     4801 tttaagggca ccaataactg ccttaaaaaa attacgcccc gccctgccac tcatcgcagt
     4861 actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg gcatgatgaa
     4921 cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatggtga
     4981 aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca
     5041 cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg aaataggcca
     5101 ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt
     5161 cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac
     5221 aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata cggaattccg
     5281 gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat
     5341 ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac
     5401 attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa
     5461 cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg
     5521 ataactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag ttggaacctc
     5581 ttacgtgccg atcaacgtct cattttcgcc aaaagttggc ccagggcttc ccggtatcaa
     5641 cagggacacc aggatttatt tattctgcga agtgatcttc cgtcacaggt atttattcgg
     5701 cgcaaagtgc gtcgggtgat gctgccaact tactgattta gtgtatgatg gtgtttttga
     5761 ggtgctccag tggcttctgt ttctatcagc tgtccctcct gttcagctac tgacggggtg
     5821 gtgcgtaacg gcaaaagcac cgccggacat cagcgctagc ggagtgtata ctggcttact
     5881 atgttggcac tgatgagggt gtcagtgaag tgcttcatgt ggcaggagaa aaaaggctgc
     5941 accggtgcgt cagcagaata tgtgatacag gatatattcc gcttcctcgc tcactgactc
     6001 gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg gcggagattt
     6061 cctggaagat gccaggaaga tacttaacag ggaagtgaga gggccgcggc aaagccgttt
     6121 ttccataggc tccgcccccc tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg
     6181 cgaaacccga caggactata aagataccag gcgtttcccc ctggcggctc cctcgtgcgc
     6241 tctcctgttc ctgcctttcg gtttaccggt gtcattccgc tgttatggcc gcgtttgtct
     6301 cattccacgc ctgacactca gttccgggta ggcagttcgc tccaagctgg actgtatgca
     6361 cgaacccccc gttcagtccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6421 cccggaaaga catgcaaaag caccactggc agcagccact ggtaattgat ttagaggagt
     6481 tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt ggtgactgcg
     6541 ctcctccaag ccagttacct cggttcaaag agttggtagc tcagagaacc ttcgaaaaac
     6601 cgccctgcaa ggcggttttt tcgttttcag agcaagagat tacgcgcaga ccaaaacgat
     6661 ctcaagaaga tcatcttatt aatcagataa aatatttcta gatttcagtg caatttatct
     6721 cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgt
//
