LOCUS       sequence-467856-        6810 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET-28a(+) based plasmid for expression of H16_A1358 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 6810)
  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 6810)
  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: pSLB025
FEATURES             Location/Qualifiers
     source          1..6810
                     /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             1675..1692
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1745..1763)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1759..1806
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1843..2298
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1930..1949)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2140..2161
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2390..3205)
                     /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(2390..3205)
                     /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     3113..3132
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3197..3216
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3327..3915
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3816..3835
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4069..4086
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4101..4240
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4166..4185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4326..4348
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4342..4533)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5342..6424)
                     /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     6386..6405
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6425..6502)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6708..6727
                     /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 atgtacaagg agagaatccg tcttgctcgc
      181 cttcatgata aggtcgtaag cgcggacgaa gcggccgcgc tcattcgtga cggaatgaca
      241 gttggaatgt ccgggttcac gcgggcaggt gactgtaaag aggttccctt tgcattggcg
      301 cgtagagcag ctacagaacc actccgtatc acattgatga ccggtgcctc actgggtaat
      361 gacattgata gagtactggc tgaggccgac gtcatcgcgc gccgtttacc gttccaaagc
      421 gatgcgaccc tgcgccggaa aatcaatgca ggcgaagtca tgttcataga ccaacattta
      481 tctgagacgg ttgagcaact tcgctcaggc caaatcgcgc cagtggatgt cgccgttgtc
      541 gaggcagttg cgataactga gcaaggtggc atcataccta gcacgtcggt cggtaactcc
      601 gcttcattcg ctattttagc gcgcaaggtc attgtcgaga tcaacctcaa catgcccctg
      661 gaattggaag ggctgcacga tatttatttc ccggtgcgtc gtccatatcg gcagcctatt
      721 ccgcttattg ctcccgaaca gcgtattcgg ttaccctaca tccccatcga tccagagaag
      781 atagctgcga tagtaatcac tgctaaggac gacagtccat caaacgctct gcccccggat
      841 gccgatacac gtaatatcgc cggtcacttg aacgacttcc tgttacgtga ggttcgtgca
      901 ggccgcttgt caccttcgct tcaaccatta caagcaggaa ttggtaccat agcaaacgct
      961 gtcctgcatg ggatggtaga tagcccgttc cgtgatctgc aaatgtacag cgaagtgctg
     1021 caggatagca cgatagaatt actggacgct ggcagattgg cgtttgcgag tgcttcaagt
     1081 gtgacactga cgcgcgaggt ctaccagaga ttcctgagta acctggatag atatcgctcc
     1141 agactgttac ttcgaccgca agaaataagt aaccaccctg agatcttacg gcgactgggt
     1201 ttgatcacta tcaataccgc actggagtgc gacatctacg ggaacgtaaa cagcacgcac
     1261 gtcggtggca cccacatgat gaacggaatc ggcggcagcg gggacttcgc ccgcaacgcc
     1321 cacttatccg tatttgtaac caagtccgtg gctaaggatg gggatgcatc atctattgtt
     1381 ccgatggtag cccatgtgga tcatacggaa cacgacgtgg atatcatcgt cactgagcac
     1441 gggttagcgg atctccgcgg tcttgctcct cgggagcggg cccggacggt catcgacaac
     1501 tgtgcggacc cgcaatatcg ggaattatta ggagattact tcagacgtgc cagcactcac
     1561 ggtgggcaaa ctccccacct gttggaagaa gcactttcct ggcatgttgg attccgcgac
     1621 agaggcacca tgctgcctgc acagcctgct caagcaatgg ccgcgtaact cgagcaccac
     1681 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     1741 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1801 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     1861 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1921 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1981 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2041 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2101 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2161 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2221 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     2281 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2341 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     2401 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     2461 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     2521 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     2581 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     2641 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     2701 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     2761 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     2821 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     2881 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     2941 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     3001 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     3061 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     3121 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     3181 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     3241 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3301 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3361 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3421 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3481 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3541 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3601 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3661 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3721 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3781 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3841 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3901 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3961 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4021 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4081 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4141 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4201 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4261 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4321 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4381 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     4441 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     4501 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     4561 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     4621 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     4681 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     4741 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4801 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     4861 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     4921 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     4981 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     5041 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     5101 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     5161 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     5221 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     5281 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     5341 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5401 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     5461 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     5521 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     5581 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     5641 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     5701 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     5761 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     5821 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     5881 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     5941 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     6001 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     6061 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     6121 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     6181 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     6241 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     6301 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     6361 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     6421 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     6481 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     6541 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     6601 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     6661 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     6721 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     6781 tagaggatcg agatctcgat cccgcgaaat
//
