LOCUS       sequence-467863-        6981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET-28a(+) based plasmid for expression of H16_B1335 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 6981)
  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 6981)
  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: pSLB030
FEATURES             Location/Qualifiers
     source          1..6981
                     /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             1846..1863
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1916..1934)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1930..1977
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2014..2469
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2101..2120)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2311..2332
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2561..3376)
                     /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(2561..3376)
                     /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     3284..3303
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3368..3387
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3498..4086
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3987..4006
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4240..4257
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4272..4411
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4337..4356)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4497..4519
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4513..4704)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5513..6595)
                     /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     6557..6576
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6596..6673)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6879..6898
                     /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 atgacacgcc ccaccccaat tcaccgtaca
      181 ccaagtgcct atgcctatcc gttattgatc aaacaattgc ttcacgcacc ccttgccgtc
      241 aatcctgaac aagaaattgt ctacagaggc caagtacgcc acagctattg gaccatgcga
      301 caccgcattg gtcaactcgc ttctggcttg caatccctcg ggattgcgca aggggacacg
      361 gttgccgtca tggattggga cagccaccgg tacttagagt catacttcgg gattcccatg
      421 atgggagcag tattgatgac agtgaatgtc cgcttaagcc cagagcaaat tgcgtacaca
      481 ctgaatcatg cgggtgtgcg cctgctcatg gtacacgccg attttcttcc ggttgtcgca
      541 gcaatccgcg atcaactgga gtcggtcgaa tgtttagttt tgatcgggga cgacactctc
      601 tctctggagt cggcaccact tgctgagggc tttgtcgcag agtacgaaac ccttctggcg
      661 gcttccccgc ctgattaccc ttttccagac tttgacgaga acgcccgagc caccacattc
      721 tacaccacgg ggacaactgg tctgcctaaa ggggtctact tttcccatcg acagattgtt
      781 ttgcacacct tggcaacaat ggcggcgtta accagtgcac cgacgcaagg tagagtacac
      841 agagacgatg tctatatgcc acttacccca atgttccatg tgcatgcctg gggtatgcct
      901 ttcgtggcta cagctctggg tatgaagcaa gtctaccctg gtcgttatgc tgccgaccag
      961 ctccttcagt tagtgcaatc tgagcggatc acgttcagcc acggcgtccc gactctgtta
     1021 aatatgatat taagccaccc tgactctgcg gcggttgact tatcaggcat gaaaatcata
     1081 gtcggcggtt cggcattacc ccgcggactg gctcaggcag ctattgaccg tggcattgat
     1141 gttttcactg gctacggtat gtcggagact tgccctatac tgactttggc acaagtcaaa
     1201 acgtcactct taggtaacgc cactactgag cttgacgttc gtaccaagac gggacttcct
     1261 gtgcctctcg tcgatcttag aatcgttgat gatgaaatgc gcgacatagc gcacgacgga
     1321 aaggcttccg gagagattgt ggtgcgtgca ccttggctta ctcaaggtta cttcgggaat
     1381 gcagcggaga gtgagcacct gtgggcaggc ggatacttgc ataccaatga cataggctcc
     1441 attgacagtg acggttatct ccaggtgacg gatcgtatca aggatgtcat taagagtggt
     1501 ggcgaatggg tttcctctct tgagttggaa gacctgatat cgcgccacgc ggccgtatca
     1561 gaggtagccg ttataggtat taaggatgct cgctggggtg aacgcccact gcctttaatt
     1621 gtccttcggc caggccaaag cgcaggcgcc gaggaaattc aagagcactt acgcgacttt
     1681 gtgcgccgcg gagctatctc aaagtatgcc gtacccgagc gtgtgttatt tgtagaggcg
     1741 attgagaaga catcggttgg taagattaac aagcgcttgt taagagacaa ataccaagcc
     1801 gcacccccac caagtcctaa ttctgggacg cacccgtaac tcgagcacca ccaccaccac
     1861 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     1921 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     1981 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     2041 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2101 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2161 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2221 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2281 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2341 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     2401 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     2461 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2521 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     2581 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     2641 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     2701 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     2761 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     2821 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     2881 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     2941 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     3001 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     3061 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     3121 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     3181 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     3241 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     3301 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     3361 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     3421 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3481 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3541 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     3601 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     3661 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     3721 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     3781 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     3841 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     3901 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     3961 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4021 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4081 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4141 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4201 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4261 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4321 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     4381 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     4441 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     4501 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     4561 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     4621 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     4681 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     4741 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     4801 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     4861 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     4921 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     4981 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     5041 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     5101 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     5161 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     5221 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     5281 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     5341 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     5401 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     5461 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     5521 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5581 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     5641 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     5701 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     5761 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     5821 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     5881 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     5941 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     6001 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     6061 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     6121 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     6181 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     6241 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     6301 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     6361 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     6421 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     6481 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     6541 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     6601 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     6661 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     6721 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     6781 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     6841 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     6901 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     6961 gagatctcga tcccgcgaaa t
//
