LOCUS       sequence-324904-        5792 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NLUC in pET-28 a (+) backbone for bacterial IPTG inducible 
            expression (KmR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5792)
  AUTHORS   Urquiza-Garcia U, Millar AJ
  TITLE     Expanding the bioluminescent reporter toolkit for plant science with
            NanoLUC.
  JOURNAL   Plant Methods. 2019 Jul 8;15:68. doi: 10.1186/s13007-019-0454-4. 
            eCollection 2019.
  PUBMED    31316580
REFERENCE   2  (bases 1 to 5792)
  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: "Plant 
            Methods."; date: "2019-07-8"; volume: "15:68. doi"; pages: " 
            10.1186/s13007-019-0454-4. eCollection 2019"
COMMENT     Sequence Label: pET-28a(+)::NL
FEATURES             Location/Qualifiers
     source          1..5792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(211..726)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     RBS             733..755
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    770..794
                     /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(794..813)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(795..813)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(897..916)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1122..1199
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1200..2282
                     /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     complement(1219..1238)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3091..3282
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3276..3298)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3384..3523
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3439..3458
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3538..3555)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3709..4297)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3789..3808)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4408..4427)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4419..5234
                     /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             4419..5234
                     /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     complement(4492..4511)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5326..5781)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5463..5484)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5675..5694
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccggtcacc ttacgccaga atgcgttcgc acagccgcca
      241 gccggtcact ccgttgatgg ttactcggaa cagcagggag ccgtcggggt tgatcaggcg
      301 ctcgtcgata attttgttgc cgttccacag ggtccctgtt acagtgatct ttttgccgtc
      361 gaacacggcg atgccttcat acggccgtcc gaaatagtcg atcatgttcg gcgtaacccc
      421 gtcgattacc agtgtgccat agtgcaggat caccttaaag tgatgatcat ccacagggta
      481 caccacctta aaaatttttt cgatctggcc catttggtcg ccgctcagac cttcatacgg
      541 gatgatgaca tggatgtcga tcttcagccc attttcaccg ctcaggacaa tcctttggat
      601 cggagttacg gacaccccga gattctgaaa caaactggac acacctccct gttcaaggac
      661 ttggtccagg ttgtagccgg ctgtctgtcg ccagtcccca acgaaatctt cgagtgtgaa
      721 gaccatggta tatctccttc ttaaagttaa acaaaattat ttctagaggg gaattgttat
      781 ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga tctcgatcct
      841 ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta
      901 tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg
      961 tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt
     1021 gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt
     1081 cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc gaatggcgca
     1141 aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg
     1201 tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt
     1261 cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg
     1321 cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt
     1381 cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg
     1441 cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac
     1501 gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg
     1561 ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca
     1621 ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt
     1681 tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc
     1741 aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct
     1801 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
     1861 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
     1921 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
     1981 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
     2041 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
     2101 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
     2161 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
     2221 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     2281 gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg
     2341 cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc
     2401 gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc
     2461 tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt
     2521 gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa
     2581 cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc
     2641 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
     2701 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
     2761 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     2821 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     2881 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     2941 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     3001 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     3061 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     3121 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     3181 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     3241 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     3301 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     3361 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     3421 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg
     3481 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     3541 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3601 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3661 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3721 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3781 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3841 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3901 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3961 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4021 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4081 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4141 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4201 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4261 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4321 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa
     4381 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa
     4441 cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     4501 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     4561 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     4621 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     4681 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc
     4741 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     4801 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     4861 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     4921 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     4981 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     5041 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     5101 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     5161 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     5221 atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5281 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     5341 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     5401 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     5461 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     5521 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     5581 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     5641 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     5701 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     5761 atgcgccgct acagggcgcg tcccattcgc ca
//
