LOCUS       sequence-461106-        5789 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of L. plantarum larE and LarC genes in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5789)
  AUTHORS   Nevarez JL, Turmo A, Gatreddi S, Gupta S, Hu J, Hausinger RP
  TITLE     Overcoming barriers for investigating nickel-pincer nucleotide 
            cofactor-related enzymes.
  JOURNAL   mBio. 2024 Dec 16:e0340424. doi: 10.1128/mbio.03404-24.
  PUBMED    39679682
REFERENCE   2  (bases 1 to 5789)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "mBio. 2024 
            Dec 16:e0340424. doi: 10.1128/mbio.03404-24."
COMMENT     Sequence Label: pRSFDuet-larE-larC
FEATURES             Location/Qualifiers
     source          1..5789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(482..1564)
                     /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     1526..1545
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1565..1642)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1688..1706
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1707..1731
                     /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)."
     promoter        2657..2675
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2676..2700
                     /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)."
     CDS             4030..4074
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4112..4130)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4126..4173
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4403..5218)
                     /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(4403..5218)
                     /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     5126..5145
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5219..5310)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5326..5789,1..286))
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(5336..5353)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5576..5595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
       61 cattggtaac tgatttagag gactttgtct tgaagttatg cacctgttaa ggctaaactg
      121 aaagaacaga ttttggtgag tgcggtcctc caacccactt accttggttc aaagagttgg
      181 tagctcagcg aaccttgaga aaaccaccgt tggtagcggt ggtttttctt tatttatgag
      241 atgatgaatc aatcggtcta tcaagtcaac gaacagctat tccgttactc tagatttcag
      301 tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgtaat
      361 tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
      421 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
      481 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
      541 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
      601 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
      661 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
      721 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
      781 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
      841 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
      901 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
      961 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     1021 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     1081 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     1141 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     1201 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     1261 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     1321 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     1381 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     1441 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     1501 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     1561 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     1621 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     1681 aggaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctgtagaa
     1741 ataattttgt ttaactttaa taaggagata taccatggca acattagcaa caaaaaaagc
     1801 aacgttagta gcagcattaa aggatttaca acgggtcacc gtggcctttt ctggtgggat
     1861 cgacagtacc ttagttttga agatggccct cgatgtttta ggccgtgaca atgtgaccgc
     1921 tgtggttgcg aactctgaat tatttacgga tgaagaattc gataaagcga tgagtttagc
     1981 cgaagaacta ggtgctaacg ttcaaggaac tacgctagac tacttgagcg atgaccacat
     2041 caagaacaac acgcctgata gctggtacta tgcgaagaag atgttctaca gccggttgaa
     2101 tgacattgcg gctaataacg gtagtgccgc tgtcttagac ggtatgatca agaatgatga
     2161 aaatgattat cgtcctggtt tgaaggcccg tagtgaagcg ggggcccgga gcttgttaca
     2221 agaagctgat ttcttcaaga ctgacgttcg ggccttggca caagaactcg gtttgacgaa
     2281 ctggaataaa gtggcgtcat gctctgtatc ttcacggttc ccatacggga cgacgttgac
     2341 acatgacaat attgctcaag taatggccgc cgaaaaatat ttgcgtagtt taggttttcc
     2401 aacggtccgg gttcgtttcc acaatgatat tgcgcggatc gaattaccag aagcgcgaat
     2461 tggtgatttc ttagtcttta acgaccgcgt taaccgtcaa ttacaatcac ttggattccg
     2521 ctacgttacc cttgatttag gtggcttccg tagtggtcgg atgaatgata cgttgaccaa
     2581 ggcacaattg gccactttcg cctagcttaa gtcgaacaga aagtaatcgt attgtacacg
     2641 gccgcataat cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
     2701 ccatcttagt atattagtta agtataagaa ggagatatac atatgggtgc tcaaacactt
     2761 tatttagacg ctttttcggg gattagcggt gatatgtttc tgggcgcgtt actcgattta
     2821 ggacttgatt ttgagcaatt aaaaaccgaa ttggctaagt tacacgtgca cggttatgaa
     2881 ttaacgcagc aacgggaagc ccagagtagc atctatggca cgagctttga tgttcaggtt
     2941 gcgggtggta aggaccatgg ttttgtggaa catcaccatc atcagcatga agctgggcat
     3001 catcatgatc atgaagcgcg tcatttagct gatattgagg cgttaattga cggcagtgac
     3061 ttatcagata ccgtgaaaca ccacgcgaaa gcgatcttta tggaaattgc gcaagcggaa
     3121 gcagcggttc atcacatgcc actagctgaa gttcatttcc atgaagtggg cgcgttggat
     3181 tcgattgttg atattgtggg ctgctgtatt gggttggaat tgatgcagat tgataccatc
     3241 atggcttcac cgttaagtga tggtagtggt tttattaacg ttgcccatgg tcagatgcca
     3301 gttcccgtac cagccgtgat gcagatgcga gttggtagtg ccattccgat tcaacaacgg
     3361 ttggatgtgc atactgaatt gattaccccc actgggatgg gcttggtcaa gacgctcgta
     3421 cgtgaattcg gaccgttacc ggaaaatgcc gtgccaacta gagttggtta tgggtttggt
     3481 aaacgggaca cgggtggttt caatgcactg cgagccgtat tattcgaaaa aaagaaacta
     3541 agtcagcaaa tcgttaaccg gacggctgat gctgtcttaa tgattgaagc taatctggat
     3601 gatcagacgg gtgaaggact ggggtacgtg atgaaccagt tattaacggc gggcgcatac
     3661 gatgtgtttt tcacgcccat tcagatgaag aaagatcggc cggcgacaaa gttaacggtt
     3721 ttgggaaacg ttaacgataa ggacttactg actaagttga ttttacaaga aacgacgacg
     3781 attggtgtac gttaccagac gtggcaacgt actattatgc aacggcactt cctgacggtc
     3841 gcaacgccgt acggtgacgt tcaagttaaa gtggcgacat atcaagatat tgagaagaag
     3901 atgccggaat atgcggattg tgcccagctg gcgcagcaat ttcatattcc gtttagaacg
     3961 gtctaccagg cagccttagt ggcggtagat caattagatg aggaggcgta aggtaccctc
     4021 gagtctggta aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact
     4081 agcgcagctt aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt
     4141 ggggcctcta aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac
     4201 acggtcacac tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct
     4261 atttaacgac cctgccctga accgacgaca agctgacgac cgggtctccg caagtggcac
     4321 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     4381 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     4441 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     4501 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     4561 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     4621 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     4681 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     4741 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcggtcg ctgttaaaag
     4801 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     4861 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     4921 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     4981 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     5041 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     5101 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     5161 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcatac
     5221 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5281 tatttgaatg tatttagaaa aataaacaaa taggcatgca gcgctcttcc gcttcctcgc
     5341 tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggtgtcagct cactcaaaag
     5401 cggtaatacg gttatccaca gaatcagggg ataaagccgg aaagaacatg tgagcaaaaa
     5461 gcaaagcacc ggaagaagcc aacgccgcag gcgtttttcc ataggctccg cccccctgac
     5521 gagcatcaca aaaatcgacg ctcaagccag aggtggcgaa acccgacagg actataaaga
     5581 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5641 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5701 tgttggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5761 cccgttcagc ccgaccgctg cgccttatc
//
