LOCUS       sequence-229027-        8185 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon optimized CarCBADE enzymes from Pectobacterium carotovorum in 
            pBbaA5k backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8185)
  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 8185)
  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: pCarCBADE
FEATURES             Location/Qualifiers
     source          1..8185
                     /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      4663..4734
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4750..4777
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(4935..5523)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5015..5034)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5611..5705)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5736..6530)
                     /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(5847..5866)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6457..6476
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(6715..7797)
                     /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     7759..7778
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7798..7875)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8081..8100
                     /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 tgaggatcta ggaggaaaaa aatgcctaac gatctttacg
     3241 ctatctacaa tcgttacacg tcaaggaccc tgttttttaa atactgtgcg acagcgactc
     3301 taacgcacag actgacacgt cggctgtcgc tttttacgtt gaaaaagtgc cttgcccgtc
     3361 cacagggacg ccttttctct ctggttaata gcatttattt tggcggagaa acgctggaag
     3421 aggttcagag tacagcaact tttctggcac gctctggcat tgcttgcgta ctggactacg
     3481 ccgtggaagg ggaaaatgac gagacgcagt tcgataaggc aatggaaaat acgctgcgcc
     3541 ttatcgagat gtcgcagcaa acggatagcc tgccttttgt cgtgattaaa ccgtcgtctc
     3601 tgggtagtgt tgctgtgtat gctcggcaaa gtgaaaggct cgcgctggat gaagcatctg
     3661 caagtgcatg gtcacgcatc gttacgcgtt tctcacgtct gttcgattat gcccgctctc
     3721 atggcgtgca tgtgatggtt gatgcagagc agaccgcgat tcagcccgca gtggatcgtt
     3781 tggttctaga catgatgcgt gaatttaacc gcgatagcgc ggtgatcacg ctgacgctgc
     3841 aattttatct aaaggatcaa ttgcgttttc tcgacgaatg ctatcagcga gcctgtcagg
     3901 ataatttcct gttcggcgtg aaagtagtgc gcggtgctta tctggaagaa gagaaacgag
     3961 tgaacggtgg cgtacgctgt tttgcaacca aacaggaaac cgatcgtagt tataacgcgg
     4021 cggtggatta cattgcactg cgtctggatc gaatcgcacc gtttttcgcc acgcacaacg
     4081 aggaaagcct cgctttgatt atgtcgagtg agtcgctacg cgcggggcgc acctgggtag
     4141 gccagcttta cgggttgggc gatcatatta cttattcgct gctgcaaact ggtttccgcg
     4201 tgtgcaaata cctgccttac ggtccgctcg ataagtcgtt gccctattta ctccgcagga
     4261 ttgaagagaa cgcggttgcc tcggcaacct tcaaaaagga aaataaactc ttgcagaagg
     4321 agttgctgcg ccgtctcgta ggagggatgt aatgtcaaaa tttcattgtc atattcgcga
     4381 tacggctatc ggtttccccg tgtcggagga tgaaagcgta ctcagttcag cctatgaagc
     4441 tggtgtcgaa ctgccttatc gctgtgcttc tggttactgt ggtgtatgca aggtgcgcct
     4501 gacgtccggc aacgtcaaca tggatcattc tggtgggatc tcacgtaaag atatcgctga
     4561 tggttacatt ctaccttgct gttctgtgcc gctctctaac ctcgaaattg aacctgtatc
     4621 gtcatgctga ggatccaaac tcgagtaagg atctccaggc atcaaataaa acgaaaggct
     4681 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag
     4741 agtcacactg gctcaccttc gggtgggcct ttctgcgttt atacctaggg cgttcggctg
     4801 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4861 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4921 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4981 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5041 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5101 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5161 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5221 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5281 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5341 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     5401 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5461 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5521 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5581 taagggattt tggtcatgac tagtgcttgg attctcacca ataaaaaacg cccggcggca
     5641 accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag
     5701 gagtccaagc gagctctcga accccagagt cccgctcaga agaactcgtc aagaaggcga
     5761 tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca
     5821 gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag
     5881 cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc
     5941 atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg
     6001 cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga
     6061 tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc
     6121 gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca
     6181 gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc
     6241 acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg
     6301 caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc
     6361 agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg
     6421 aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc
     6481 tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat
     6541 cctcatcctg tctcttgatc agatcatgat cccctgcgcc atcagatcct tggcggcaag
     6601 aaagccatcc agtttacttt gcagggcttc ccaaccttac cagagggcgc cccagctggc
     6661 aattccgacg tcggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     6721 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     6781 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     6841 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     6901 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     6961 gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga
     7021 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     7081 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     7141 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     7201 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     7261 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     7321 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     7381 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     7441 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     7501 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     7561 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     7621 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     7681 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     7741 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     7801 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     7861 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     7921 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     7981 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     8041 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     8101 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     8161 tcgagatcgt ttaggcaccc caggc
//
