LOCUS       sequence-467838-        6966 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET-28a(+) based plasmid for expression of H16_A3514 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 6966)
  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 6966)
  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: pSLB026
FEATURES             Location/Qualifiers
     source          1..6966
                     /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             1831..1848
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1901..1919)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1915..1962
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1999..2454
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2086..2105)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2296..2317
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2546..3361)
                     /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(2546..3361)
                     /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     3269..3288
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3353..3372
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3483..4071
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3972..3991
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4225..4242
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4257..4396
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4322..4341)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4482..4504
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4498..4689)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5498..6580)
                     /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     6542..6561
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6581..6658)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6864..6883
                     /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 atgacgcggc cagtgacccc aggactttcc
      181 gtagtagccg gttctaccga cataccgctg agtgacgaaa ccatcccagc acttttggcg
      241 agaacagtag ccgccaaccc aggagccccg gctgtggtat tccgcgagca cggcgttaga
      301 tggagctggg ctgagttctc agatcaggtt gatagattag ctagcgggct tttgagacga
      361 ggcattcagc ctggcgaccg tgtaggtata tgggcgccca atcggccaga gtggttggtc
      421 actcaatttg ccactgcccg gataggtgct atcttggtga acattaaccc ggcttatcgc
      481 cgagcggagt tggaatacgc tttaaacaag gctggtgtcc gaatgcttat aacagctgcc
      541 cgtttcaagg cttccgatta cttagcaacc ctgcaggcat tggcccctga gcttgcggaa
      601 gcagagcccg gtgctttacg gagcgcccgt cttccacgat tgcagtgggt gatccggatg
      661 ggtggtgagg ctactcctgg gatgctcaac tacgacgctc ttttagcaga gcctgacaca
      721 gtgcggcttg atgctgtgac agcaacattg agcgcattgg acgccatcaa catccagttt
      781 acatccggta ccacgggtaa tcccaagggt gctactctta cacatcacaa cgtagttaat
      841 aatggacgct tcgttgcaat agcgatgaga ctcggggatt ctgacgtcct gtgcattcct
      901 gttccactct accactgctt tggcatggta cttagcgtac ttgcgtgcgt atccgtcgga
      961 gcttgtatgg ttttcccagg cgaggcgttt gatcccttgg ctaccatgca agccgcatct
     1021 gaggaacgct gcactgcttt acatggcgtg cccacaatgt tcatcgcaca gcttgaccac
     1081 cccgagttct cacgattcga cttttctgca ttgcgtaccg ggataatggc gggtgctccg
     1141 tgtcctatag aggtaatgaa gcgcgtggtg gctgatatgc acatgagtga ggtgactatc
     1201 gcttacggca tgactgagac ctcacctgta tcattccaat cggcgacaga cgaccctctc
     1261 gacaaacgtg tagcaacagt gggcagagtc caacctcatt tagagtgcaa agtcgtcgat
     1321 gccctgggag aagttgttcc gactggggct accggcgagt tgtgcacacg aggctatagt
     1381 gttatgcaag gctattggga agacgacgaa cgaacccgtg aagcaatcag agacgggtgg
     1441 atgcacaccg gagacctggc gacaattgat gacgagggct actgcaatat agttgggcgc
     1501 gtcaaagaca tgttgatccg tggtggtgag aacatatatc cacgcgaaat tgaggagttt
     1561 cttttccgtc acccgaaagt gcaggcggtt caggtcttcg gtgtcccgga ccagaagtat
     1621 ggagaggaag tatgcgcgtg gatagtactg aaaccaggcc aatctgcaac agaagaggaa
     1681 attcgtaagt tctgccggga ccaaatagct cattacaaga tcccccgtta tatacggttt
     1741 gtgtcggaga tgcctatgac tgtaacagga aaagtgcaga agttcgtgat gcgagacaca
     1801 atgatccacg acttggggct ctgactcgag caccaccacc accaccactg agatccggct
     1861 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     1921 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     1981 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2041 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2101 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2161 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2221 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2281 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2341 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2401 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     2461 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2521 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     2581 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     2641 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     2701 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     2761 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     2821 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     2881 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     2941 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     3001 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     3061 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     3121 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     3181 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     3241 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     3301 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     3361 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     3421 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3481 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3541 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3601 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     3661 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     3721 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3781 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3841 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3901 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3961 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4021 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4081 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4141 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4201 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     4261 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     4321 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     4381 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4441 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4501 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     4561 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     4621 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     4681 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     4741 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     4801 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     4861 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     4921 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     4981 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     5041 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     5101 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     5161 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     5221 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     5281 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     5341 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     5401 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     5461 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5521 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5581 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     5641 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     5701 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     5761 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     5821 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     5881 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     5941 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     6001 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     6061 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     6121 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     6181 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     6241 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     6301 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     6361 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     6421 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     6481 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     6541 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     6601 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     6661 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     6721 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     6781 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     6841 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     6901 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     6961 cgaaat
//
