LOCUS       sequence-467923-        7251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET-28a(+) based plasmid for expression of H16_A0285 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 7251)
  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 7251)
  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: pSLB024
FEATURES             Location/Qualifiers
     source          1..7251
                     /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             2116..2133
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2186..2204)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2200..2247
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2284..2739
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2371..2390)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2581..2602
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2831..3646)
                     /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(2831..3646)
                     /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     3554..3573
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3638..3657
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3768..4356
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4257..4276
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4510..4527
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4542..4681
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4607..4626)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4767..4789
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4783..4974)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5783..6865)
                     /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     6827..6846
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6866..6943)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7149..7168
                     /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 atgcaagaat catcagctac aacgttccct
      181 cgctggcttt tagcccacgc tcagcagcgt cctgaccatc cggcacatcg ggagaaggat
      241 ttgggcatct ggcagactta tagctgggca caagcagctc agcaggtgcg agcgcttgcg
      301 tgcggcctgg ccgcgctcgg tttccgaaga gggatgaatt tagcagtagt tggggacaac
      361 agaccaagac tgtactgggc catgactgca gcccaggctc tgggtggtgt gcccgtgccc
      421 ttgtatcaag acgccatcgc aaatgaaatg gtctacgtcc tgaacgacgc agagatagaa
      481 tttgcaattg ttgaggatca agaacaggtg gataaattgt tagaggtcga agctcaactc
      541 gccgagtcag gccgcgctgt ccggcacgtc attttcgagg accctcgtgg cctgatggac
      601 tatgatcacc caagtctgat gagctacgag caattacaag agttgggccg cgagttcgat
      661 caagcacatc ccgggttcta tgatgaagcg atagcggccg gtcaaccgga tgacacagcg
      721 ataatattat acacctcggg aactacgggt aagcctaagg gagtgtgcca ctcacactac
      781 ggtttaatcg gtgcagcccg caacggctgc gccttcgata aattgtctgc ggacgacgat
      841 gtgctcagct atcttccaat ggcatgggtt ggcgacaatc tgtttagtta tgcgcaggca
      901 atggtcgctg gcttcacggt taactgtcct gagtcacggg aaaccgtgat gacagactta
      961 cgcgagattg ggccgacgta ctactttgcg ccaccacgta tctacgaagg tctgctgacg
     1021 caagttatga ttcgcatgga agatgcgggt tggataaaac gcaagctttt ccactgggct
     1081 atggatgtgg ccaagcggtg tggcactgac atcctggatg gaaaacctgt gtcattggct
     1141 gaccgggctc ggtacgcatt gggagaggct cttgtatacg gacccttacg caatgtgctc
     1201 ggtatgagcc gtatacgggt ggggtatact gctggcgagg cgatcggtcc agacctgttt
     1261 cgcttttaca gaagcattgg agtgaacctt aaacaattct acggacaaac cgaaacctgt
     1321 gcctatgttt gtctgcaacc agatggtaag gttaagtttg attctgttgg gccggcggcg
     1381 ccgggtatgg aaattcgcat agctgacaac ggagaagttt tagttcgtgg agtcgggctg
     1441 ttgaagagct attacaagag agatgatgcg acgcgtgagg ccatcaacga cgagggctac
     1501 tttatgacgg gcgacgctgg cgttatagat gctgacggtc acctcaagat tatcgatcgt
     1561 gccaaggacg ttggcaaact ggcggacgga tctatgtttg ctccgaagta catcgagaat
     1621 aagctgaagt tcttccctta catcaaagaa gccgtagcat tcggaaatga tcgtgatcag
     1681 gtctgcgcct tcatcaacat tgacttcgag gcggttggta attgggcaga gcgtcgtcac
     1741 ctgccgtatg ccggttacgt cgacttagca gctcagccag aagttctgga aatgattgga
     1801 gaatgcgtca accaagtcaa cgctgacttg gcaaatgacc caatgttagc gggcagccaa
     1861 gtggcgcgct ttctcgtcct tcataaggaa ctggacccag acgacgacga gctcacccgg
     1921 acacggaagg tacgccgcgg atttattgcc gacaagtatg gtgtgttagt ggaagccctc
     1981 tacgccggaa agagcgagca attcattgag acgcgagtta aatttgagga cggacgcgaa
     2041 gggtctgttt ctgcaacttt gaaattggtg aatgcaaaac gcttaccagt ggctgcacgc
     2101 gcagcctaac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     2161 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     2221 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     2281 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2341 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2401 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2461 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2521 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2581 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2641 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2701 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     2761 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2821 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     2881 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     2941 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     3001 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     3061 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     3121 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     3181 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     3241 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     3301 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     3361 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     3421 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     3481 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     3541 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     3601 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     3661 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     3721 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3781 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3841 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3901 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3961 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4021 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4081 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4141 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4201 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4261 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4321 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4381 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4441 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4501 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4561 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     4621 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     4681 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4741 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4801 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     4861 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     4921 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     4981 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     5041 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     5101 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     5161 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     5221 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     5281 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     5341 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     5401 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     5461 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     5521 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     5581 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     5641 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     5701 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     5761 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5821 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     5881 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     5941 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     6001 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     6061 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     6121 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     6181 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     6241 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     6301 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     6361 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     6421 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     6481 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     6541 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     6601 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     6661 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     6721 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     6781 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     6841 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     6901 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     6961 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     7021 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     7081 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     7141 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     7201 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa t
//
