LOCUS       sequence-467864-        6945 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET-28a(+) based plasmid for expression of H16_B1102 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 6945)
  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 6945)
  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: pSLB027
FEATURES             Location/Qualifiers
     source          1..6945
                     /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             1810..1827
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1880..1898)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1894..1941
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1978..2433
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2065..2084)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2275..2296
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2525..3340)
                     /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(2525..3340)
                     /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     3248..3267
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3332..3351
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3462..4050
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3951..3970
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4204..4221
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4236..4375
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4301..4320)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4461..4483
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4477..4668)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5477..6559)
                     /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     6521..6540
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6560..6637)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6843..6862
                     /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 atgcgtgatt acgcccaagc attcgacggc
      181 ttttcctatg acgatgccgt cgcacggcaa ctgcacggct cccaagaggc tatgaacgca
      241 tgcgttgagt gttgcgaccg tcacgcgttg cctggccgga ttgcactgtt ctgggaaggc
      301 cgcgatggta acagccggtc ctggactttc accgagttac aggctttgtc cgcacagttt
      361 gctggttttc tgaaggcgca aggagtacaa ccaggggata gagttgccgg cctgctgccg
      421 cgtaatgcag aacttctggt gactatcttg ggaacgtggc gggcaggggc ggtgtaccaa
      481 cccttattta cagctttcgg gcccaaggcc attgagcacc ggctgaacgc atcgggcgca
      541 aaagtggtcg ttacagatgg cgccaaccgc cctaagttgg acgacgtcga tggttgtccg
      601 gcgatcgtca ccgttgccgg cgacaaagga cgtggactgg tcagaggtga cttctctttc
      661 tgggcggaac tggaaagaca gccggcctcg tttgaacccg tgccgagacg cggtgatgat
      721 cctttcttaa tgatgttcac aagtggaacc accgggcctg ccaagccttt gctggttccg
      781 ttgaaggcca tagccgcctt tgccgggtac atgtctgatg cagttgacct tcgcgcagaa
      841 gacgcctttt ggaatttggc agaccccggc tgggcttacg gtctttacta cgctgtgacc
      901 gggccgttgg cgctcggtca ccctaccacg ttctacgacg gccccttcac tgtagagagt
      961 acttgcagag taattcgaaa gtacggcatc acaaatctcg ccggttcacc gaccgcctat
     1021 cgactgctta tcgccgccgg tgaagccgtc tccggtccgt tacgtggtcg attgcgagct
     1081 gttagcagcg caggcgagcc gttaaatccc gaggtcattc gctggttcgc aagtgagctg
     1141 ggcgtaacga tccacgacca ctacggccaa actgagttgg gtatggtact gtgtaatcac
     1201 cacgccctgg cccatcctgt tcgcatgggc gcagcgggat ttgcctcgcc ggggcatcga
     1261 gttgtggttg tcgacgacga acagagagaa ctcccaccgg gtcggcctgg aacactggct
     1321 ttggacctca aacggtctcc aatgtgctgg ttcggtgggt accacggcac gcccacgagt
     1381 ggatttgcgg gtgggtacta tttgacaggg gactcggcgg agctcaatga cgacggttca
     1441 atctctttca tcggccgggc agacgacgtc atcacgacga gcggttatcg cgttggtccc
     1501 tttgacgtgg agtccgcctt aatcgagcat cctgccgttg tagaggccgc tgtaataggt
     1561 aagcctgacc ctgaaagaac ggagttaata aaggcattcg ttgtgcttga cccacaatac
     1621 agagcggcgc ctgaattagc agaggctctg cggcagcatg ttcggaagcg tttggctgca
     1681 catgcatacc cgcgggaaat tgaatttgtg gtggagctgc ccaaaacgcc cagtggcaag
     1741 gtacagcgtt ttattctccg gaaccaagaa gttgcgcgtg cgcgcgaggc cgccgctgcg
     1801 tgactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     1861 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1921 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     1981 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2041 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2101 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2161 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2221 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2281 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2341 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2401 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     2461 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     2521 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     2581 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     2641 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     2701 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     2761 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     2821 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     2881 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     2941 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     3001 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     3061 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     3121 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     3181 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     3241 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     3301 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     3361 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     3421 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     3481 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     3541 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     3601 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     3661 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     3721 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3781 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3841 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3901 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3961 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4021 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4081 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4141 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4201 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     4261 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     4321 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     4381 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4441 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4501 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     4561 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     4621 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     4681 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     4741 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     4801 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     4861 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     4921 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     4981 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     5041 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     5101 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     5161 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     5221 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     5281 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     5341 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     5401 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     5461 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5521 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     5581 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     5641 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     5701 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     5761 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     5821 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     5881 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     5941 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     6001 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     6061 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     6121 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     6181 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     6241 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     6301 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     6361 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     6421 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     6481 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     6541 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     6601 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     6661 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     6721 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     6781 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     6841 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     6901 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaat
//
