LOCUS       sequence-222193-        6758 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon optimized CarCBA enzymes from Pectobacterium carotovorum in 
            pBbaA5k backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6758)
  AUTHORS   Shomar H, Gontier S, van den Broek NJF, Tejeda Mora H, Noga MJ, 
            Hagedoorn PL, Bokinsky G
  TITLE     Metabolic engineering of a carbapenem antibiotic synthesis pathway 
            in Escherichia coli.
  JOURNAL   Nat Chem Biol. 2018 Aug;14(8):794-800. doi: 
            10.1038/s41589-018-0084-6. Epub 2018 Jun 25.
  PUBMED    29942079
REFERENCE   2  (bases 1 to 6758)
  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: 
            "10.1038/s41589-018-0084-6"; journalName: "Nat Chem Biol"; date: 
            "2018-08"; volume: "14"; issue: "8"; pages: "794-800"
COMMENT     Sequence Label: pCarCBA
FEATURES             Location/Qualifiers
     source          1..6758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(49..1131)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     1093..1112
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1132..1209)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1415..1434
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(1520..1525,1526..1543,1544..1550)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    1558..1574
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     terminator      4755..4826
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4842..4869
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(5027..5615)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5107..5126)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5703..5797)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5828..6622)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5939..5958)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6549..6568
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gacgtcggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
       61 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
      121 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
      181 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
      241 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
      301 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt
      361 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
      421 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
      481 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
      541 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
      601 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
      661 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
      721 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
      781 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
      841 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
      901 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
      961 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     1021 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     1081 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     1141 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     1201 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     1261 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     1321 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     1381 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     1441 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     1501 tcgtttaggc accccaggct ttacacttta tgcttccggc tcgtataatg tgtggaattg
     1561 tgagcggata acaatttcag aattcaaaag atctaggagg aaaaaaatgt ccgagatcgt
     1621 gaaatttaac cctgtgatgg cttctggctt cggcgcatac attgatcacc gtgacttcct
     1681 ggaagctaaa acggaaacga tcaaaaacct gctgatgcgc caaggtttcg tggttgtcaa
     1741 aaacctggat attgactccg atactttccg tgatatctac tctgcatacg gtaccatcgt
     1801 cgaatacgcg gatgaaaaaa ttggtgttgg ttttggttat cgcgataccc tgaaactgga
     1861 aggcgaaaaa ggtaaaatcg ttaccggtcg tggtcaactg cctttccacg ctgacggtgg
     1921 tctgctgctg tctcaggtcg atcaggtttt cctgtacgcg gcggagatta aaaacgtcaa
     1981 attccgcggt gcaactaccg tttgcgatca cgctctggcg tgccaggaaa tgccagcaca
     2041 cctgctgcgc gttctggaag aggagacgtt cgaagttcgt gtgctggaac gtggttacta
     2101 cgtggatgtg tcccctgatg gttggttcaa agtgccggtg ttcactgatc tgggttgggt
     2161 tcgtaaaatg ctgatctatt tcccattcga tgaaggccag ccggcaagct gggaaccgcg
     2221 tattgttggt tttaccgacc acgaaaccca ggcattcttt caagaactgg gtgcgttcct
     2281 gaaacagccg cgttattact acaaacattt ctgggaagac ggtgacctgc tgattatgga
     2341 taaccgtcgt gttatccacg agcgtgaaga atttaacgat gatgacatcg ttcgtcgtct
     2401 gtaccgcggc caaactgcag atatctaaag gaggaaaaaa tggtattcga agaaaactcc
     2461 gatgaagtac gcgtgattac cctggatcac ccgaacaaac ataacccatt ttcccgtacc
     2521 ctggaaacta gcgtgaagga cgctctggct cgtgcaaacg cggatgatag cgtgcgtgcg
     2581 gtcgttgtgt atggcggcgc tgaacgtagc ttcagcgcag gcggtgattt caacgaagtc
     2641 aagcagctgt ctcgctccga agatattgaa gaatggatcg atcgtgtgat cgacctgtat
     2701 caggcagtcc tgaacgtaaa caaaccgacg atcgcggccg ttgatggcta cgcgatcggt
     2761 atgggcttcc aattcgctct gatgtttgac cagcgcctga tggccagcac cgcgaacttc
     2821 gtgatgccgg aactgaaaca cggtatcggt tgctctgtag gtgcagcaat cctgggtttc
     2881 acccatggtt tctccaccat gcaagaaatc atctaccagt gccagtctct ggatgcaccg
     2941 cgctgcgtcg actaccgcct ggtgaatcag gtcgtagagt cttccgcgct gctggacgcg
     3001 gcgatcactc aggcgcacgt gatggcgtcc tacccggctt ccgcattcat taacacgaag
     3061 cgtgcggtaa acaagccgtt cattcacctg ctggaacaga cccgtgatgc ttccaaagca
     3121 gtgcacaaag ccgcattcca ggcgcgtgat gcccagggtc acttcaaaaa cgttctgggt
     3181 aagaaatact gaggatctag gaggaaaaaa atgagcaata gcttctgcgt cgtttacaaa
     3241 ggttccgata ctgacattaa caacattcag cgtgatttcg acggtaaagg cgaagccctg
     3301 tctaacggct atctgttcat cgaacagaac ggccactacc agaaatgcga aatggagcgt
     3361 ggcacggcgt acctgattgg ttctctgtat aaccgtactt tcctgattgg cctggcgggc
     3421 gtttgggaag gtgaagcata cctggctaac gatgctgaac tgctggccct gctgtttacg
     3481 cgcctgggtg ccaacgcact ggctctggcg gaaggcgatt tctgcttttt cattgatgaa
     3541 ccgaatggtg agctgaccgt tatcacggaa tctcgcggct tctccccggt ccatgttgtt
     3601 caaggtaaga aggcatggat gacgaacagc ctgaaactgg taactgctgc cgaaggcgag
     3661 ggcgcactgt ggtttgaaga agaagctctg gtgtgccagt ctctgatgcg tgccgatact
     3721 tatacgccgg ttaagaacgc gcagcgtctg aaaccgggcg cagtgcacgt tctgacccac
     3781 gatagcgaag gctacagctt tgtagagtct cgtaccctga ccactccggc atccaaccaa
     3841 ctgctggctc tgccgcgtga accgctgctg gcactgatcg atcgctacct gaacgctccg
     3901 ctggaggacc tggcaccacg cttcgacacg gttggtatcc cgctgagcgg tggtctggac
     3961 tcttccctgg tgactgccct ggctagccgc catttcaaga aactgaacac gtattccatc
     4021 ggtactgagc tgtccaacga gtttgagttc agccagcagg ttgcagatgc tctgggtacg
     4081 caccaccaga tgaaaatcct gtccgaaacg gaagttatta acggtatcat cgagtccatt
     4141 tactacaacg aaatcttcga cggtctgagc gcggaaatcc agagcggtct gttcaacgtg
     4201 taccgtcagg ctcagggcca agtctcctgc atgctgactg gctatggttc tgacctgctg
     4261 ttcggcggca tcctgaaacc tggcgctcag tatgataatc cgaaccagct gctggcggaa
     4321 caagtgtacc gcacccgttg gaccggcgaa tttgcgaccc acggtgcgtc ttgctacggc
     4381 atcgatatcc gccacccgtt ttggtcccac agcctgatta gcctgtgcca cgccctgcac
     4441 cctgattaca agattttcga taatgaagtt aaaaacatcc tgcgtgaata cgctgatagc
     4501 ctgcagctgc tgccaaagga catcgtttgg cgtaaaaaga tcggtattca cgaaggttct
     4561 tctgttaatc aggcgttcgc aaacgtactg ggttctacgg ttgacaatta ccagaccaaa
     4621 tctcgtttca cttatcgtgt ttaccaggcg ttcctgcgcg gtcgcctgtc catcactgac
     4681 gttaccccgt cccagctgaa ggatctgatc aaaaaggact gaggatccaa actcgagtaa
     4741 ggatctccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     4801 tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc
     4861 ctttctgcgt ttatacctag ggcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4921 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4981 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5041 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5101 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5161 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5221 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5281 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5341 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5401 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5461 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5521 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5581 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5641 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg actagtgctt
     5701 ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa atccagatgg
     5761 agttctgagg tcattactgg atctatcaac aggagtccaa gcgagctctc gaaccccaga
     5821 gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag
     5881 cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa
     5941 tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt
     6001 cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat
     6061 gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg
     6121 ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca
     6181 tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg
     6241 gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag
     6301 caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc
     6361 ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg
     6421 atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa
     6481 aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg
     6541 tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt
     6601 gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga tcagatcatg
     6661 atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact ttgcagggct
     6721 tcccaacctt accagagggc gccccagctg gcaattcc
//
