LOCUS       sequence-229023-        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"
     promoter        join(1..6,7..24,25..31)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    39..55
                     /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      3236..3307
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3323..3350
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(3508..4096)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3588..3607)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4184..4278)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4309..5103)
                     /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(4420..4439)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5030..5049
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(5288..6370)
                     /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     6332..6351
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6371..6448)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6654..6673
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tttacacttt atgcttccgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
       61 gaattcaaaa gatctaggag gaaaaaaatg tccgagatcg tgaaatttaa ccctgtgatg
      121 gcttctggct tcggcgcata cattgatcac cgtgacttcc tggaagctaa aacggaaacg
      181 atcaaaaacc tgctgatgcg ccaaggtttc gtggttgtca aaaacctgga tattgactcc
      241 gatactttcc gtgatatcta ctctgcatac ggtaccatcg tcgaatacgc ggatgaaaaa
      301 attggtgttg gttttggtta tcgcgatacc ctgaaactgg aaggcgaaaa aggtaaaatc
      361 gttaccggtc gtggtcaact gcctttccac gctgacggtg gtctgctgct gtctcaggtc
      421 gatcaggttt tcctgtacgc ggcggagatt aaaaacgtca aattccgcgg tgcaactacc
      481 gtttgcgatc acgctctggc gtgccaggaa atgccagcac acctgctgcg cgttctggaa
      541 gaggagacgt tcgaagttcg tgtgctggaa cgtggttact acgtggatgt gtcccctgat
      601 ggttggttca aagtgccggt gttcactgat ctgggttggg ttcgtaaaat gctgatctat
      661 ttcccattcg atgaaggcca gccggcaagc tgggaaccgc gtattgttgg ttttaccgac
      721 cacgaaaccc aggcattctt tcaagaactg ggtgcgttcc tgaaacagcc gcgttattac
      781 tacaaacatt tctgggaaga cggtgacctg ctgattatgg ataaccgtcg tgttatccac
      841 gagcgtgaag aatttaacga tgatgacatc gttcgtcgtc tgtaccgcgg ccaaactgca
      901 gatatctaaa ggaggaaaaa atggtattcg aagaaaactc cgatgaagta cgcgtgatta
      961 ccctggatca cccgaacaaa cataacccat tttcccgtac cctggaaact agcgtgaagg
     1021 acgctctggc tcgtgcaaac gcggatgata gcgtgcgtgc ggtcgttgtg tatggcggcg
     1081 ctgaacgtag cttcagcgca ggcggtgatt tcaacgaagt caagcagctg tctcgctccg
     1141 aagatattga agaatggatc gatcgtgtga tcgacctgta tcaggcagtc ctgaacgtaa
     1201 acaaaccgac gatcgcggcc gttgatggct acgcgatcgg tatgggcttc caattcgctc
     1261 tgatgtttga ccagcgcctg atggccagca ccgcgaactt cgtgatgccg gaactgaaac
     1321 acggtatcgg ttgctctgta ggtgcagcaa tcctgggttt cacccatggt ttctccacca
     1381 tgcaagaaat catctaccag tgccagtctc tggatgcacc gcgctgcgtc gactaccgcc
     1441 tggtgaatca ggtcgtagag tcttccgcgc tgctggacgc ggcgatcact caggcgcacg
     1501 tgatggcgtc ctacccggct tccgcattca ttaacacgaa gcgtgcggta aacaagccgt
     1561 tcattcacct gctggaacag acccgtgatg cttccaaagc agtgcacaaa gccgcattcc
     1621 aggcgcgtga tgcccagggt cacttcaaaa acgttctggg taagaaatac tgaggatcta
     1681 ggaggaaaaa aatgagcaat agcttctgcg tcgtttacaa aggttccgat actgacatta
     1741 acaacattca gcgtgatttc gacggtaaag gcgaagccct gtctaacggc tatctgttca
     1801 tcgaacagaa cggccactac cagaaatgcg aaatggagcg tggcacggcg tacctgattg
     1861 gttctctgta taaccgtact ttcctgattg gcctggcggg cgtttgggaa ggtgaagcat
     1921 acctggctaa cgatgctgaa ctgctggccc tgctgtttac gcgcctgggt gccaacgcac
     1981 tggctctggc ggaaggcgat ttctgctttt tcattgatga accgaatggt gagctgaccg
     2041 ttatcacgga atctcgcggc ttctccccgg tccatgttgt tcaaggtaag aaggcatgga
     2101 tgacgaacag cctgaaactg gtaactgctg ccgaaggcga gggcgcactg tggtttgaag
     2161 aagaagctct ggtgtgccag tctctgatgc gtgccgatac ttatacgccg gttaagaacg
     2221 cgcagcgtct gaaaccgggc gcagtgcacg ttctgaccca cgatagcgaa ggctacagct
     2281 ttgtagagtc tcgtaccctg accactccgg catccaacca actgctggct ctgccgcgtg
     2341 aaccgctgct ggcactgatc gatcgctacc tgaacgctcc gctggaggac ctggcaccac
     2401 gcttcgacac ggttggtatc ccgctgagcg gtggtctgga ctcttccctg gtgactgccc
     2461 tggctagccg ccatttcaag aaactgaaca cgtattccat cggtactgag ctgtccaacg
     2521 agtttgagtt cagccagcag gttgcagatg ctctgggtac gcaccaccag atgaaaatcc
     2581 tgtccgaaac ggaagttatt aacggtatca tcgagtccat ttactacaac gaaatcttcg
     2641 acggtctgag cgcggaaatc cagagcggtc tgttcaacgt gtaccgtcag gctcagggcc
     2701 aagtctcctg catgctgact ggctatggtt ctgacctgct gttcggcggc atcctgaaac
     2761 ctggcgctca gtatgataat ccgaaccagc tgctggcgga acaagtgtac cgcacccgtt
     2821 ggaccggcga atttgcgacc cacggtgcgt cttgctacgg catcgatatc cgccacccgt
     2881 tttggtccca cagcctgatt agcctgtgcc acgccctgca ccctgattac aagattttcg
     2941 ataatgaagt taaaaacatc ctgcgtgaat acgctgatag cctgcagctg ctgccaaagg
     3001 acatcgtttg gcgtaaaaag atcggtattc acgaaggttc ttctgttaat caggcgttcg
     3061 caaacgtact gggttctacg gttgacaatt accagaccaa atctcgtttc acttatcgtg
     3121 tttaccaggc gttcctgcgc ggtcgcctgt ccatcactga cgttaccccg tcccagctga
     3181 aggatctgat caaaaaggac tgaggatcca aactcgagta aggatctcca ggcatcaaat
     3241 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     3301 cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttataccta
     3361 gggcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3421 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3481 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3541 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3601 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3661 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3721 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3781 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3841 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3901 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     3961 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4021 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4081 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4141 cgaaaactca cgttaaggga ttttggtcat gactagtgct tggattctca ccaataaaaa
     4201 acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgag gtcattactg
     4261 gatctatcaa caggagtcca agcgagctct cgaaccccag agtcccgctc agaagaactc
     4321 gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac
     4381 gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc
     4441 tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg
     4501 gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc
     4561 gccgtcgggc atgcgcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg
     4621 ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc
     4681 gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg
     4741 ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag
     4801 atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc
     4861 gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc
     4921 ctgcagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg
     4981 cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata
     5041 gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat
     5101 catgcgaaac gatcctcatc ctgtctcttg atcagatcat gatcccctgc gccatcagat
     5161 ccttggcggc aagaaagcca tccagtttac tttgcagggc ttcccaacct taccagaggg
     5221 cgccccagct ggcaattccg acgtcggtgc ctaatgagtg agctaactta cattaattgc
     5281 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5341 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     5401 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     5461 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     5521 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa
     5581 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     5641 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     5701 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     5761 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     5821 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     5881 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     5941 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     6001 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     6061 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     6121 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     6181 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     6241 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     6301 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     6361 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     6421 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     6481 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     6541 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     6601 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     6661 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     6721 ccggcgtaga ggatcgagat cgtttaggca ccccaggc
//
