LOCUS       sequence-467860-        6870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET-28a(+) based plasmid for expression of H16_B1264 from 
            Cupriavidus necator H16, with N-terminal hexahistidine tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6870)
  AUTHORS   Holmes EC, Breunig SL, Johnson CW, Beckham GT, Bleem AC
  TITLE     Insights into genetic determinants of volatile fatty acid catabolism
            in Cupriavidus necator H16.
  JOURNAL   Appl Environ Microbiol. 2025 Jun 12:e0051525. doi: 
            10.1128/aem.00515-25.
  PUBMED    40503888
REFERENCE   2  (bases 1 to 6870)
  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: "Appl 
            Environ Microbiol. 2025 Jun 12:e0051525. doi: 10.1128/aem.00515-25."
COMMENT     Sequence Label: pSLB029
FEATURES             Location/Qualifiers
     source          1..6870
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1735..1752
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1805..1823)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1819..1866
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1903..2358
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1990..2009)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2200..2221
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2450..3265)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2450..3265)
                     /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
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3173..3192
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3257..3276
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3387..3975
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3876..3895
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4129..4146
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4161..4300
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4226..4245)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4386..4408
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4402..4593)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5402..6484)
                     /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     6446..6465
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6485..6562)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6768..6787
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg atgacaagag gaatcacttt gggagatacg
      181 ttgagttggc ccgcacgtta ccttcctcgc aagacagctt tagtgtccgg aatcggtgcc
      241 ggacgtcgtg cctggactta cgctgaatta gacgccgagg tcaaccggca cgctcacgcg
      301 ctgcaatcat tgggaatcgg caagggagac gttgtcgccg ccttcttgta caataccccg
      361 gcgttcgtat tcactcttct tgcgaccgcc cggatcggtg ctgtttacaa ccccgtcaat
      421 taccggctcg cagctcagga gctcgcatac atattaaacg acggtggtgc caaagccgtt
      481 ttatttgaac aggaaggggc aggagtggtc gagaaggcag ctgaactggc gcctggtact
      541 gcgttacgta tttacgcaga cccagacgca gctcccgcat tcgccaccca ccgcttagac
      601 acgttggctg cagcacaacc agccacccca ccagcagtaa cagttcacga ggacgaccct
      661 tgcattttaa tgtatacgtc aggcactacg gggcgtccaa agggtgttgt tcacactcac
      721 cgaagtaagc tgcagcacaa cgccatgatg caccaagcga tgacgttgag cagagaggac
      781 gttggtctgt ctgttgcccc gctgaaccat accgccgagt tgcatacgag cttcttacca
      841 cgccttcaat taggagctac ccaggtttta caacgacgct ttgatgcggg cgaggcatgg
      901 caattggtcg aaagtgaacg ggtgactcac ttcttcgcgg cgcctacgat ggtcggaatg
      961 ctgctgaatc acccggatgc tgacgcacgg gatgtatctt ccttgcgttt agtagaatac
     1021 ggtggcgcca gcatggctcc gcatttgata agagagtggg accgaaaggt cggtgccggc
     1081 ttagtgcagg tgtacggcac taccgagatg ggcccgtgta tgtcagtatt gtacccccat
     1141 gaacagttgt ctcatgctgg ctccgccgga ctgccggcac tgggacacga gctggtcgtg
     1201 gcacgcttac gtgacgacgg agaacctacc gaccctatgc aaccatgtgt acctggcgag
     1261 gtaggtgagg tcttagttcg cggtccatgt atgatgcaag gatacctgaa ccgcccggac
     1321 gctaatgcac gggcactcgc tcacggctgg tatcacactg gtgaccttgg ttctctggac
     1381 gccgacggct acctctggat ccgtgaccgg atcgactata tgataaactc gggagcagag
     1441 aacgtatacc cgcgcgaggt tgaggacgca ctgatcgaac atccggctgt gctggaagtt
     1501 gctgtgcttg gcgaaccaga cccgacgtgg ggtcaagtag tcggcgccta tgtcgtaacc
     1561 cgaggggacg gcccggtcag tgctgcccaa cttgacgcgt ttctcttaca gggtgaccgg
     1621 ttagcggctt acaagcgccc gcgccgctac cacttcgtcg acgcactccc caaaaccacc
     1681 agtggcaaga ttcagaagca catgctgcgg gctggagcga cggcgtaact cgagcaccac
     1741 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     1801 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1861 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     1921 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1981 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2041 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2101 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2161 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2221 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2281 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     2341 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2401 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     2461 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     2521 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     2581 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     2641 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     2701 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     2761 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     2821 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     2881 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     2941 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     3001 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     3061 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     3121 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     3181 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     3241 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     3301 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3361 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3421 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3481 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3541 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3601 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3661 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3721 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3781 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3841 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3901 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3961 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4021 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4081 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4141 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4201 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4261 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4321 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4381 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4441 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     4501 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     4561 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     4621 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     4681 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     4741 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     4801 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4861 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     4921 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     4981 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     5041 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     5101 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     5161 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     5221 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     5281 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     5341 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     5401 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5461 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     5521 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     5581 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     5641 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     5701 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     5761 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     5821 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     5881 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     5941 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     6001 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     6061 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     6121 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     6181 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     6241 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     6301 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     6361 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     6421 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     6481 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     6541 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     6601 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     6661 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     6721 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     6781 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     6841 tagaggatcg agatctcgat cccgcgaaat
//
