LOCUS       sequence-386868-        7597 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MoClo Level 1 luciferase reporter: NOS::TMV::LucN::flag::nosT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7597)
  AUTHORS   Cai YM, Witham S, Patron NJ
  TITLE     Tuning Plant Promoters Using a Simple Split Luciferase Method to 
            Assess Transcription Factor-DNA Interactions.
  JOURNAL   ACS Synth Biol. 2023 Nov 17;12(11):3482-3486. doi: 
            10.1021/acssynbio.3c00094. Epub 2023 Oct 19.
  PUBMED    37856867
REFERENCE   2  (bases 1 to 7597)
  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"; doi: 
            "10.1021/acssynbio.3c00094"; journalName: "ACS Synth Biol"; date: 
            "2023-11-17- 17"; volume: "12"; issue: "11"; pages: "3482-3486"
COMMENT     Sequence Label: pEPSW1KN0035
FEATURES             Location/Qualifiers
     source          1..7597
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(281..1096)
                     /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(281..1096)
                     /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     1004..1023
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    1529..1553
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        1656..1835
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     misc_feature    1871..1926
                     /label=TMV Omega
                     /note="translational enhancer from the tobacco mosaic virus
                     5'-leader sequence (Gallie et al., 1988)"
     CDS             1928..2440
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             2501..2524
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      2540..2792
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    2816..2840
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             4141..4770
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             5199..6272
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      6338..6532
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(6768..6790)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6876..7016
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6931..6950
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(7031..7048)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(7202..7597,1..193))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7282..7301)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
       61 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
      121 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
      181 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
      241 aaactcacgt taagggattt tggtcatgca ttctaggtga ttagaaaaac tcatcgagca
      301 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
      361 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
      421 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
      481 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
      541 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
      601 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agtcgaaata
      661 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
      721 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
      781 ctgttttccc tgggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
      841 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
      901 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
      961 tcccatacaa tcggtagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     1021 acccatataa atcagcatcc atgttggaat ttaatcgcgg ccttgagcaa gacgtttccc
     1081 gttgaatatg gctcataaca gaacttatta tttccttcct cttttctaca gtatttaaag
     1141 ataccccaag aagctaatta taacaagacg aactccaatt cactgttcct tgcattctaa
     1201 aaccttaaat accagaaaac agctttttca aagttgtttt caaagttggc gtataacata
     1261 gtatcgacgg agccgatttt gaaaccgcgg tgatcacagg cagcaacgct ctgtcatcgt
     1321 tacaatcaac atgctaccct ccgcgagatc atccgtgttt caaacccggc agcttagttg
     1381 ccgttcttcc gaatagcatc ggtaacatga gcaaagtctg ccgccttaca acggctctcc
     1441 cgctgacgcc gtcccggact gatgggctgc ctgtatcgag tggtgatttt gtgccgagct
     1501 gccggtcggg gagctgttgg ctggctggtg gcaggatata ttgtggtgta aacataacga
     1561 attgctcttc aatgaggaga gcggagaatt aagggagtca cgttatgacc cccgccgatg
     1621 acgcgggaca agccgtttta cgtttggaac tgacagaacc gcaacgttga aggagccact
     1681 cagccgcggg tttctggagt ttaatgagct aagcacatac gtcagaaacc attattgcgc
     1741 gttcaaaagt cgcctaaggt cactatcagc tagcaaatat ttcttgtcaa aaatgctcca
     1801 ctgacgttcc ataaattccc ctcggtatcc aattagagtc tcatattcac tctcatactg
     1861 tatttttaca acaattacca acaacaacaa acaacaaaca acattacaat tactatttac
     1921 aattacaatg gtattcacac tcgaagattt cgttggggac tggcgacaga cagccggcta
     1981 caacctggac caagtccttg aacagggagg tgtgtccagt ttgtttcaga atctcggggt
     2041 gtccgtaact ccgatccaaa ggattgtcct gagcggtgaa aatgggctga agatcgacat
     2101 ccatgtcatc atcccgtatg aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat
     2161 ttttaaggtg gtgtaccctg tggatgatca tcactttaag gtgatcctgc actatggcac
     2221 actggtaatc gacggggtta cgccgaacat gatcgactat ttcggacggc cgtatgaagg
     2281 catcgccgtg ttcgacggca aaaagatcac tgtaacaggg accctgtgga acggcaacaa
     2341 aattatcgac gagcgcctga tcaaccccga cggctccctg ctgttccgag taaccatcaa
     2401 cggagtgacc ggctggcggc tgtgcgaacg cattctggct tcggattata aggaccatga
     2461 cggagactat aaggaccatg acctcgacgc tgcagcagcg gattataagg acgatgacga
     2521 taagtgagct tgtcaagcag atcgttcaaa catttggcaa taaagtttct taagattgaa
     2581 tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt
     2641 aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc
     2701 gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt
     2761 atcgcgcgcg gtgtcatcta tgttactaga tcgacgctag catgaagagc aattctgaca
     2821 ggatatattg gcgggtaaac ctaagagaaa agagcgttta ttagaataat cggatattta
     2881 aaagggcgtg aaaaggttta tccgttcgtc catttgtatg tgcatgccaa ccacagggtt
     2941 cccctcggga tcaaagtact ttgatccaac ccctccgctg ctatagtgca gtcggcttct
     3001 gacgttcagt gcagccgtca tctgaaaacg acatgtcgca caagtcctaa gttacgcgac
     3061 aggctgccgc cctgcccttt tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt
     3121 agaatacttg cgactagaac cggagacatt acgccatgaa caagagcgcc gccgctggcc
     3181 tgctgggcta tgcccgcgtc agcaccgacg accaggactt gaccaaccaa cgggccgaac
     3241 tgcacgcggc cggctgcacc aagctgtttt ccgagaagat caccggcacc aggcgcgacc
     3301 gcccggagct ggccaggatg cttgaccacc tacgccctgg cgacgttgtg acagtgacca
     3361 ggctagaccg cctggcccgc agcacccgcg acctactgga cattgccgag cgcatccagg
     3421 aggccggcgc gggcctgcgt agcctggcag agccgtgggc cgacaccacc acgccggccg
     3481 gccgcatggt gttgaccgtg ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg
     3541 accgcacccg gagcgggcgc gaggccgcca aggcccgagg cgtgaagttt ggcccccgcc
     3601 ctaccctcac cccggcacag atcgcgcacg cccgcgagct gatcgaccag gaaggccgca
     3661 ccgtgaaaga ggcggctgca ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg
     3721 agcgcagcga ggaagtgacg cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg
     3781 cattgaccga ggccgacgcc ctggcggccg ccgagaatga acgccaagag gaacaagcat
     3841 gaaaccgcac caggacggcc aggacgaacc gtttttcatt accgaagaga tcgaggcgga
     3901 gatgatcgcg gccgggtacg tgttcgagcc gcccgcgcac ctctcaaccg tgcggctgca
     3961 tgaaatcctg gccggtttgt ctgatgccaa gctggcggcc tggccggcca gcttggccgc
     4021 tgaagaaacc gagcgccgcc gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg
     4081 tcatgcggtc gctgcgtata tgatgcgatg agtaaataaa caaatacgca aggggaacgc
     4141 atgaaggtta tcgctgtact taaccagaaa ggcgggtcag gcaagacgac catcgcaacc
     4201 catctagccc gcgccctgca actcgccggg gccgatgttc tgttagtcga ttccgatccc
     4261 cagggcagtg cccgcgattg ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc
     4321 atcgaccgcc cgacgattga ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg
     4381 atcgacggag cgccccaggc ggcggacttg gctgtgtccg cgatcaaggc agccgacttc
     4441 gtgctgattc cggtgcagcc aagcccttac gacatatggg ccaccgccga cctggtggag
     4501 ctggttaagc agcgcattga ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg
     4561 cgggcgatca aaggcacgcg catcggcggt gaggttgccg aggcgctggc cgggtacgag
     4621 ctgcccattc ttgagtcccg tatcacgcag cgcgtgagct acccaggcac tgccgccgcc
     4681 ggcacaaccg ttcttgaatc agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg
     4741 gccgctgaaa ttaaatcaaa actcatttga gttaatgagg taaagagaaa atgagcaaaa
     4801 gcacaaacac gctaagtgcc ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg
     4861 ccagcctggc agacacgcca gccatgaagc gggtcaactt tcagttgccg gcggaggatc
     4921 acaccaagct gaagatgtac gcggtacgcc aaggcaagac cattaccgag ctgctatctg
     4981 aatacatcgc gcagctacca gagtaaatga gcaaatgaat aaatgagtag atgaatttta
     5041 gcggctaaag gaggcggcat ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc
     5101 cccatgtgtg gaggaacggg cggttggcca ggcgtaagcg gctgggttgc ctgccggccc
     5161 tgcaatggca ctggaacccc caagcccgag gaatcggcgt gagcggtcgc aaaccatccg
     5221 gcccggtaca aatcggcgcg gcgctgggtg atgacctggt ggagaagttg aaggccgcgc
     5281 aggccgccca gcggcaacgc atcgaggcag aagcacgccc cggtgaatcg tggcaagcgg
     5341 ccgctgatcg aatccgcaaa gaatcccggc aaccgccggc agccggtgcg ccgtcgatta
     5401 ggaagccgcc caagggcgac gagcaaccag attttttcgt tccgatgctc tatgacgtgg
     5461 gcacccgcga tagtcgcagc atcatggacg tggccgtttt ccgtctgtcg aagcgtgacc
     5521 gacgagctgg cgaggtgatc cgctacgagc ttccagacgg gcacgtagag gtttccgcag
     5581 ggccggccgg catggccagt gtgtgggatt acgacctggt actgatggcg gtttcccatc
     5641 taaccgaatc catgaaccga taccgggaag ggaagggaga caagcccggc cgcgtgttcc
     5701 gtccacacgt tgcggacgta ctcaagttct gccggcgagc cgatggcgga aagcagaaag
     5761 acgacctggt agaaacctgc attcggttaa acaccacgca cgttgccatg cagcgtacga
     5821 agaaggccaa gaacggccgc ctggtgacgg tatccgaggg tgaagccttg attagccgct
     5881 acaagatcgt aaagagcgaa accgggcggc cggagtacat cgagatcgag ctagctgatt
     5941 ggatgtaccg cgagatcaca gaaggcaaga acccggacgt gctgacggtt caccccgatt
     6001 actttttgat cgatcccggc atcggccgtt ttctctaccg cctggcacgc cgcgccgcag
     6061 gcaaggcaga agccagatgg ttgttcaaga cgatctacga acgcagtggc agcgccggag
     6121 agttcaagaa gttctgtttc accgtgcgca agctgatcgg gtcaaatgac ctgccggagt
     6181 acgatttgaa ggaggaggcg gggcaggctg gcccgatcct agtcatgcgc taccgcaacc
     6241 tgatcgaggg cgaagcatcc gccggttcct aatgtacgga gcagatgcta gggcaaattg
     6301 ccctagcagg ggaaaaaggt cgaaaaggac tctttcctgt ggatagcacg tacattggga
     6361 acccaaagcc gtacattggg aaccggaacc cgtacattgg gaacccaaag ccgtacattg
     6421 ggaaccggtc acacatgtaa gtgactgata taaaagagaa aaaaggcgat ttttccgcct
     6481 aaaactcttt aaaacttatt aaaactctta aaacccgcct ggcctgtgca taactgtctg
     6541 gccagcgcac agccgaagtg ctgcaaaaag cgcctaccct tcggtcgctg cgctccctac
     6601 gccccgccgc ttcgcgtcgg cctatcgcgg ccgctggccg ctcaaaaatg gctggcctac
     6661 ggccaggcaa tctaccaggg cgcggacaag ccgcgccgtc gccactcgac cgccggcgcc
     6721 cacatcaagg caccctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     6781 cagctcccgg tgacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     6841 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     6901 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     6961 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     7021 catccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     7081 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     7141 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     7201 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     7261 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     7321 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     7381 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     7441 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     7501 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     7561 gtaacaggat tagcagagcg aggtatgtag gcggtgc
//
