LOCUS       sequence-462862-        7241 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of L. plantarum larA-strep and larB genes in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7241)
  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 7241)
  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: pETDuet-larA-strep-larB
FEATURES             Location/Qualifiers
     source          1..7241
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    733..872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(798..817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     958..980
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(974..1165)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1737..2819)
                     /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     2781..2800
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2820..2897)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3103..3122
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3210..3228
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3229..3253
                     /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             3268..3290
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4578..4601
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        4656..4674
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4675..4699
                     /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)."
     primer_bind     complement(5498..5516)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5512..5559
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5596..6051
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5683..5702)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5893..5914
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(6166..6957,6958..7026))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6789..6808
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7027..7119)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(7200..7241,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
      841 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
      901 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      961 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     1021 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     1081 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     1141 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     1201 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     1261 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     1321 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     1381 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     1441 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     1501 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     1561 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     1621 tgctagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     1681 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     1741 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     1801 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     1861 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     1921 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     1981 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     2041 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     2101 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     2161 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     2221 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     2281 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     2341 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     2401 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     2461 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     2521 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     2581 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     2641 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     2701 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     2761 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     2821 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     2881 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     2941 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     3001 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     3061 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     3121 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     3181 gatcgagatc gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     3241 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgt
     3301 ccgttgcaat tgatttacca tatgacaagc gcacaattac ggctcagatt gatgatgaaa
     3361 attacgccgg taagttagtg tcgcaagctg caacatatca taacaaatta tcggaacagg
     3421 aaacggtcga gaagtcgctg gataatccaa tcggctccga taagctggag gaacttgctc
     3481 gtgggaagca caatattgtg attattagtt ctgatcacac gcgcccagtt ccttcacata
     3541 ttatcacccc gatcctattg cggcggttac ggtcagtggc gcccgatgca cggattcgga
     3601 tcctcgtagc tactggtttc catcggccat caacccacga agaattggtg aataagtatg
     3661 gtgaagacat cgtcaataac gaagaaatcg tgatgcatgt ctcaaccgat gacagtagta
     3721 tggtcaagat tggccaatta ccatctggcg gcgattgcat tattaataag gtcgctgctg
     3781 aagcagattt gctaatctcc gaaggcttta tcgaatcaca tttctttgct ggtttttcag
     3841 gtggtcggaa gtccgtttta cctgggattg cttcatacaa gacgattatg gcgaaccatt
     3901 ccggcgaatt tattaactca ccgaaggccc ggaccggtaa tttaatgcat aattcgattc
     3961 ataaggatat ggtgtacgct gctcggaccg ctaaacttgc ctttattatc aatgttgttt
     4021 tagacgaaga taaaaaaatc attgggtcat ttgccggtga catggaagcc gcccataaag
     4081 tgggctgtga ctttgtcaaa gaactttcta gtgtaccagc cattgattgt gacattgcga
     4141 tttcgacgaa tggtggttat ccgcttgatc aaaatattta tcaggccgtt aaaggaatga
     4201 ccgctgctga agcaacaaac aaagaaggcg gcacgattat tatggttgcc ggtgctcgtg
     4261 atggtcacgg tggtgaaggg ttttatcaca acttagctga cgttgatgat cctaaggaat
     4321 tcctggacca agcaatcaac acgccacggc ttaaaactat tcctgaccaa tggacggccc
     4381 aaatctttgc tcgaatctta gttcatcatc acgtgatttt tgtgtcagac ctcgttgatc
     4441 ctgatttgat tacgaatatg catatggaac tagccaagac gttagatgaa gccatggaaa
     4501 aggcctacgc acgcgagggt caagccgcta aagtgacggt tattcctgat ggtttaggcg
     4561 ttattgtgaa ggctagctgg agtcatccac aatttgagaa gtagcttaag tcgaacagaa
     4621 agtaatcgta ttgtacacgg ccgcataatc gaaattaata cgactcacta taggggaatt
     4681 gtgagcggat aacaattccc catcttagta tattagttaa gtataagaag gagatataca
     4741 tatggcaacc acagcagaaa tattacaaca agtggcggca gggcaattaa gtccgacggc
     4801 ggctgcccaa cagttagaag cgggcaaaac agcggcatta ggttttgcca atgtcgattt
     4861 agatcgtcaa cgccgcaatg gctttccgga ggtcatctac ggtgctggta agacggcaac
     4921 ccaaattgtg ggtatcgtgc aagccttgtc ccaacagacg ttgccgattt taacgacccg
     4981 gttatccgca gaaaaattcg cggcactcca accagcgtta ccaacggctg tctatcatgc
     5041 cactgcgcag tgtatgacgg tcggggaaca acccgcaccg aaaacaccag ggtacattgc
     5101 tgtggtgacg gcggggacct ccgatcaacc ggttgctgaa gaagcagcag taaccgcgga
     5161 aacttttggc aatcgtgttg aacgagtcta tgacgtgggt gttgcgggaa tccaccgact
     5221 gtttgccaag ttggatgtga ttcgtggtgc ccgggtggtc attgtgattg cgggcatgga
     5281 aggtgcgctg gccagtgtcg ttggtggact agttgataag cccgtgattg cagtgccgac
     5341 cagcgttggt tatggtacta gttttcaagg tatgaccgcg ctattgacca tgctcaatag
     5401 ctgtgcgtcg gggattaccg ttgtcaacat tgataacggc tttggtgccg cctactcagc
     5461 tagtatggtc aatcaaatgt aacctaggct gctgccaccg ctgagcaata actagcataa
     5521 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     5581 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     5641 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     5701 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5761 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5821 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5881 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5941 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     6001 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tctggcggca
     6061 cgatggcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6121 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6181 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6241 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6301 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6361 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6421 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6481 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6541 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6601 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6661 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6721 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6781 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6841 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6901 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6961 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7021 actcatactc ttcctttttc aatcatgatt gaagcattta tcagggttat tgtctcatga
     7081 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggtcatgac caaaatccct
     7141 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     7201 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa c
//
