LOCUS       sequence-440377-        5403 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  KmR, pBTRcK broad host range vector with BTE1(C12-specific fatty 
            acid thioesterase codon-optimised for C. necator) under Ptrc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5403)
  AUTHORS   Mishra S, Perkovich PM, Mitchell WP, Venkataraman M, Pfleger B
  TITLE     Expanding the synthetic biology toolbox of Cupriavidus necator for 
            establishing fatty acid production.
  JOURNAL   J Ind Microbiol Biotechnol. 2024 Feb 16:kuae008. doi: 
            10.1093/jimb/kuae008.
  PUBMED    38366943
REFERENCE   2  (bases 1 to 5403)
  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: "J Ind 
            Microbiol Biotechnol. 2024 Feb 16:kuae008. doi: 
            10.1093/jimb/kuae008."
COMMENT     Sequence Label: pSM045
FEATURES             Location/Qualifiers
     source          1..5403
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      186..222
                     /label=soxR terminator
                     /note="bidirectional E. coli soxR transcription terminator"
     terminator      complement(336..363)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(455..541)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     694..711
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     primer_bind     complement(1677..1699)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1688..1704
                     /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)."
     promoter        complement(join(1712..1718,1719..1735,1736..1741))
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(1973..3055)
                     /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     3017..3036
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3056..3133)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     terminator      3193..3224
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(3241..4056)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3964..3983
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(4146..4811)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRRT"
     rep_origin      join(4812..5403,1..180)
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
ORIGIN
        1 aaaaaaattg atggggcaag gccgcaggcc gcgcagttgg agccggtggg tatgtggtcg
       61 aaggctgggt agccggtggg caatccctgt ggtcaagctc gtgggcaggc gcagcctgtc
      121 catcagcttg tccagcaggg ttgtccacgg gccgagcgaa gcgagccagc cggtggccgc
      181 ggaacaaaac taaagcgcca caagggcgct ttagtttgtt ttcagtccag ttacgctgga
      241 gtctgaggct cgtcctgaat gatgttattg tctcatgagc ggatacatat ttgaatgtat
      301 ttagaaaaat aaacaaaaga gtttgtagaa acgcaaaaag gccatccgtc aggatggcct
      361 tctgcttaat ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc
      421 gttgcttcgc aacgttcaaa tccgctcccg gcggatttgt cctactcagg agagcgttca
      481 ccgacaaaca acagataaaa cgaaaggccc agtctttcga ctgagccttt cgttttattt
      541 gatgcctggc agttccctac tctcgcatgg ggagacccca cactaccatc ggcgctacgg
      601 cgtttcactt ctgagttcgg catggggtca ggtgggacca ccgcgctact gccgccaggc
      661 aaattctgtt ttatcagacc gcttctgcgt tctgatttaa tctgtatcag gctgaaaatc
      721 ttctctcatc cgccaaaaca gcttattata cgcgcggctc ggcggggatg accgagatgc
      781 cgcggaacga atcggtcagt ttcggacgcc actcggtgcg ggcgcgcagc acttcgctgc
      841 cgccttccag ctgcagcaga tgatcgcaca ccaggccggc ctcgctggag ccgccgctga
      901 cggtcgtgag cgagcgcagc acggaatcgc gggtgcattc gcggcggtat tccagcgtga
      961 agctggagat atggtggctt tcgaagatcg agtctggcac cgtttcgaac acccaggcca
     1021 cgtacttgag gttgttgaca tgctggttga cgtcgaggtc attccagcgc ggcgtgaggc
     1081 cgccctggat gtagtccgcg gtggaatcgt tgagcttctg cagtttcttg atctcatcgt
     1141 ccttgaccgc cacgttgtcg atgaaggccg ggccaatctc gccgcgcacc tcatcgggga
     1201 tggtcgacag gcgccgcgtg cgcgtgttca tcagcacgct cagcgaagtg cagcgcgtca
     1261 ggatctcgcc ggtcttgcag tcgcgcacca ggaagtcgcg gcgcatgccg ttgttgcccg
     1321 acgcgccgat ccagcattcc acctcgacgg tatcgcccca ggtcggatag cgctcgacgg
     1381 ccacgtgcgt gcggcgcacc acccacatca gatcgcgctt ggacatctcc agcgtggtgc
     1441 cgaagccgtc gcccaggatg ccgaccgact tggcgtggtt gagcgtggct tcctgcatgt
     1501 ggttcatcac ggccaggatc gaggtcgagc ggtccgggcc cacttcatag gaacggatcg
     1561 cgaaggtgcg tcggaacacc aggccgtgca ggccgaagtg atcgtcgagc agctgcggca
     1621 gcttgggctt gggcttccat tccagcgtca ttattttacc tcctaatggt ctgtttcctg
     1681 tgtgaaattg ttatccgctc acaattccac acattatacg agccggatga ttaattgtca
     1741 acagctcatt tcagaatatt tgccagaacc gttatgatgt cggcgcaaaa aacattatcc
     1801 agaacgggag tgcgccttga gcgacacgaa ttatgcagtg atttacgacc tgcacagcca
     1861 taccacagct tccgatggct gcctgacgcc agaagcattg gtgcaccgtg cagtcgatga
     1921 taagctgtca aaccagatca attcgcgcta acttacatta attgcgttgc gctcactgcc
     1981 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2041 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     2101 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     2161 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     2221 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     2281 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     2341 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     2401 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     2461 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     2521 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     2581 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     2641 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     2701 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     2761 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     2821 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     2881 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     2941 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     3001 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     3061 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcacc
     3121 attcgatggt gtcaacgtaa atgcatcgct caatactgac catttaaatc atacctgacc
     3181 tccatagcag aaagtcaaaa acctccgacc ggaggctttt gacttgaggg ggatccgccc
     3241 tcagaaaaac tcatcgagca tcaagtgaaa ctgcaattta ttcatatcag gattatcaat
     3301 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     3361 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     3421 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     3481 tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca
     3541 gccattacgc tcgtcatcaa aatcactcgc accaaccaaa ccgttattca ttcgtgattg
     3601 cgcctgagcg agacgaaata cgcgatcgcc gttaaaagga caattacaaa caggaatcga
     3661 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     3721 ttcttctaat acctggaatg ctgttttccc tgggatcgca gtggtgagta accatgcatc
     3781 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     3841 tagcctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     3901 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     3961 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     4021 cctcgagcaa gacgtttccc gttgaatatg gctcatttta gcttccttag ctcctgaaaa
     4081 tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga
     4141 acctcttacg tacggcgcgg cagcgtgacc cgtgtcggcg gctccaacgg ctcgccatcg
     4201 tccagaaaac acggctcatc gggcatcggc aggcgctgct gcccgcgtcg ttcccattcc
     4261 tccgtttcgg tcaaggctgg caggtctggt tccatgcccg gaatgccggg ctggctgggc
     4321 ggctcctcgc cggggccggt cggtagttgc tgctcgcccg gatacagggt cgggatgcgg
     4381 cgcaggtcgc catgccccaa cagcgattcg tcctggtcgt cgtgatcaac caccacggcg
     4441 gcactgaaca ccgacaggcg caactggtcg cggggctggc cccacgccac gcggtcattg
     4501 accacgtagg ccgacacggt gccggggccg ttgagcttca cgacggagat ccagcgctcg
     4561 gccaccaagt ccttgactgc gtattggacc gtccgcaaag aacgtccgat gagcttggaa
     4621 agtgtcttct ggctgaccac cacggcgttc tggtggccca tctgcgccac gaggtgatgc
     4681 agcagcattg ccgccgtggg tttcctcgca ataagcccgg cccacgcctc atgcgctttg
     4741 cgttccgttt gcacccagtg accgggcttg ttcttggctt gaatgccgat ttctctggac
     4801 tgcgtggcca tgcttatctc catgcggtag gggtgccgca cggttgcggc accatgcgca
     4861 atcagctgca acttttcggc agcgcgacaa caattatgcg ttgcgtaaaa gtggcagtca
     4921 attacagatt ttctttaacc tacgcaatga gctattgcgg ggggtgccgc aatgagctgt
     4981 tgcgtacccc ccttttttaa gttgttgatt tttaagtctt tcgcatttcg ccctatatct
     5041 agttctttgg tgcccaaaga agggcacccc tgcggggttc ccccacgcct tcggcgcggc
     5101 tccccctccg gcaaaaagtg gcccctccgg ggcttgttga tcgactgcgc ggccttcggc
     5161 cttgcccaag gtggcgctgc ccccttggaa cccccgcact cgccgccgtg aggctcgggg
     5221 ggcaggcggg cgggcttcgc ccttcgactg cccccactcg cataggcttg ggtcgttcca
     5281 ggcgcgtcaa ggccaagccg ctgcgcggtc gctgcgcgag ccttgacccg ccttccactt
     5341 ggtgtccaac cggcaagcga agcgcgcagg ccgcaggccg gaggcttttc cccagagaaa
     5401 att
//
