LOCUS       sequence-469906-        6927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET-28a(+) based plasmid for expression of H16_B1148 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 6927)
  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 6927)
  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: pSLB028
FEATURES             Location/Qualifiers
     source          1..6927
                     /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             1792..1809
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1862..1880)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1876..1923
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1960..2415
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2047..2066)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2257..2278
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2507..3322)
                     /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(2507..3322)
                     /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     3230..3249
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3314..3333
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3444..4032
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3933..3952
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4186..4203
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4218..4357
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4283..4302)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4443..4465
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4459..4650)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5459..6541)
                     /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     6503..6522
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6542..6619)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6825..6844
                     /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 atgcctacgg atttcgattc gggtctggct
      181 cgtaatgcag caaatttcgt gcctctgaca ccaatcgact tccttgtcag agcggccgag
      241 gtttacggtc agcgccttgc aatagtgcac ggtccgttac gtcaaaattg gcgtgacacg
      301 tacgcgcggg cccgacggct ggccagtgca ctggcccgtg ccggtattgg aaaaggagat
      361 actgtagccg ctttgttgcc aaatacgcct gcaatggtag aggcccactt cggcgtgccg
      421 atggcgggag cagttcttaa cgcgctgaac atccgattag acgccgccaa cctggtgttc
      481 atgcttcgac atggtgaggc gcgtgttttg cttgctgata ccgaatttgc ggacctggca
      541 cgtcagatgg ctctggagat cccggggctg aaggtgatag ctgttcacga tgtcttgggt
      601 ccgcaagaag cgcctttcgg agacactgat tacgaggctt tcttggcgtc aggagattca
      661 gactacgcct ggcacatgcc cgcggatgaa tgggaagcca ttgctttgaa ctacaccagt
      721 ggaactacgg gcgaccccaa gggtgtcgtg tatcaccacc gtggtgctgc gattaacgca
      781 gtttcaaaca tactggagtg ggatttaccg aagcacccag tgtacctctg gaccttgccc
      841 atgtttcatt gtaacggttg gtgttttcct tggacagtag cggcgcgtgc tggcgtcaac
      901 gtctgccttc gcaaattcga gccgcgcttg gttttcgatc ttatgcgcga ggaaggggtt
      961 acgcactact gcgcagcccc tatcgtccat actgcgctgg tcaatgcgcc tgcagcgtgg
     1021 cgtgaaggtg ttcgtggacc tgttcgcgga atggtggcag gagcgccccc gcctgctgct
     1081 gttttagcac agatggaagc tatgggattt gagctgacac acgtgtacgg tttaacagag
     1141 gtctatggtc cggccgcggt atgcgcggag caggacgact ggtccacttt gagtgagcaa
     1201 gaccgtgctg ttcggaaagc gcgccaaggt gtacgatacc atctccaatc tcaggtcgcg
     1261 gtattagacc ccgactccat gcagccggtt ccggcggatg gagaaaccat cggcgagata
     1321 atgttccgcg ggaacatctg catgaagggg tatctgaaga acgaaaaggc tacccgtgag
     1381 gctttcgccg gcggttggtt ccacactggt gacctgggcg tctgtatgcc tgacggatat
     1441 ataaagataa aagaccgtag taaagatata atcatttctg gtggcgagaa tatctcgtca
     1501 gtggaagtcg aggacgcttt gtaccgtcat cctgccgtcc tcgcagcagc tgtggtggct
     1561 caacctgacg cacggtgggg cgagacgcca tgtgcttttg ttgaacttaa ggacggggcc
     1621 agcgtgtcgg cagaggagct gattgcacac tgccgcactt tgctggcagg cttcaaggta
     1681 cctaaggccg tttattttgg cccactgcct aagaccagca cgggcaagat tcaaaagttt
     1741 gaattgcgtc gtaaggttaa gagcgactcc gcaatcgatg tgtgactcga gcaccaccac
     1801 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     1861 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1921 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     1981 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2041 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2101 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2161 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2221 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2281 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     2341 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     2401 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2461 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     2521 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     2581 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     2641 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     2701 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     2761 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     2821 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     2881 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     2941 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     3001 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     3061 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     3121 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     3181 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     3241 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     3301 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     3361 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3421 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3481 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3541 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     3601 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3661 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3721 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3781 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3841 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3901 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3961 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4021 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4081 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4141 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4201 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4261 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     4321 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     4381 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4441 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     4501 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     4561 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     4621 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     4681 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     4741 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     4801 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     4861 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     4921 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     4981 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     5041 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     5101 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     5161 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     5221 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     5281 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     5341 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     5401 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     5461 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5521 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     5581 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     5641 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     5701 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     5761 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     5821 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     5881 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     5941 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     6001 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     6061 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     6121 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     6181 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     6241 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     6301 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     6361 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     6421 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     6481 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     6541 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     6601 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     6661 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     6721 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     6781 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     6841 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     6901 aggatcgaga tctcgatccc gcgaaat
//
