LOCUS       sequence-386865-        8531 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MoClo Level 1 luciferase reporter: proARF18::LucN::flag::notT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8531)
  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 8531)
  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: pEPSW1KN0018
FEATURES             Location/Qualifiers
     source          1..8531
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      312..506
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(742..764)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    850..990
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     905..924
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1005..1022)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1176..1764)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1256..1275)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1852..2667)
                     /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(1852..2667)
                     /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     2575..2594
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    3100..3124
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             4433..4945
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             5006..5029
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      5045..5297
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    5321..5345
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             6646..7275
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             join(7704..8531,1..246)
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="VSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
ORIGIN
        1 cgttttctct accgcctggc acgccgcgcc gcaggcaagg cagaagccag atggttgttc
       61 aagacgatct acgaacgcag tggcagcgcc ggagagttca agaagttctg tttcaccgtg
      121 cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga ggcggggcag
      181 gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc atccgccggt
      241 tcctaatgta cggagcagat gctagggcaa attgccctag caggggaaaa aggtcgaaaa
      301 ggactctttc ctgtggatag cacgtacatt gggaacccaa agccgtacat tgggaaccgg
      361 aacccgtaca ttgggaaccc aaagccgtac attgggaacc ggtcacacat gtaagtgact
      421 gatataaaag agaaaaaagg cgatttttcc gcctaaaact ctttaaaact tattaaaact
      481 cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga agtgctgcaa
      541 aaagcgccta cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg tcggcctatc
      601 gcggccgctg gccgctcaaa aatggctggc ctacggccag gcaatctacc agggcgcgga
      661 caagccgcgc cgtcgccact cgaccgccgg cgcccacatc aaggcaccct gcctcgcgcg
      721 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggtgacgg tcacagcttg
      781 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
      841 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac
      901 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
      961 agatgcgtaa ggagaaaata ccgcatcagg cgctcatccg cttcctcgct cactgactcg
     1021 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     1081 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     1141 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     1201 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     1261 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     1321 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     1381 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     1441 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     1501 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     1561 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     1621 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     1681 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     1741 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     1801 cagtggaacg aaaactcacg ttaagggatt ttggtcatgc attctaggtg attagaaaaa
     1861 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     1921 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     1981 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     2041 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     2101 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     2161 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     2221 gagtcgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     2281 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     2341 tacctggaat gctgttttcc ctgggatcgc agtggtgagt aaccatgcat catcaggagt
     2401 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     2461 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     2521 cgcatcgggc ttcccataca atcggtagat tgtcgcacct gattgcccga cattatcgcg
     2581 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gccttgagca
     2641 agacgtttcc cgttgaatat ggctcataac agaacttatt atttccttcc tcttttctac
     2701 agtatttaaa gataccccaa gaagctaatt ataacaagac gaactccaat tcactgttcc
     2761 ttgcattcta aaaccttaaa taccagaaaa cagctttttc aaagttgttt tcaaagttgg
     2821 cgtataacat agtatcgacg gagccgattt tgaaaccgcg gtgatcacag gcagcaacgc
     2881 tctgtcatcg ttacaatcaa catgctaccc tccgcgagat catccgtgtt tcaaacccgg
     2941 cagcttagtt gccgttcttc cgaatagcat cggtaacatg agcaaagtct gccgccttac
     3001 aacggctctc ccgctgacgc cgtcccggac tgatgggctg cctgtatcga gtggtgattt
     3061 tgtgccgagc tgccggtcgg ggagctgttg gctggctggt ggcaggatat attgtggtgt
     3121 aaacataacg aattgctctt caatgaggag gaaaaaaaag aatacagcat gaacaccagc
     3181 gactcagaac tgactcggca cgtggtaaca taaaactcac acgcttccca agacgctcgt
     3241 cccataaaga gtcatggctg tgaccttgtg cgagtgtggg tgtagccata gcctttgtgg
     3301 gtcctaaata aaaaaaacga tcaatggcat aatgtgtaat taactcacat tccacagtgt
     3361 taaataaaaa ttaaatctgt aacggaaaaa acaatctatc tgatgttaca caaaaaaaaa
     3421 aaataacgga aaaaagaaaa tgtggccggg gtttaataga gaagccgaca cgtttgcctg
     3481 ccacgatcca gacacaagac gatgtgggta acggcaccgt caagtgctaa cggcaacggg
     3541 gtcatcagta aaaagggcaa tttagggata tagagtaaag tcataagctt atttattact
     3601 atcgtcttga tcggacggct gagatttgcc ctctccttta aatgcaccgt acgttttttt
     3661 tttacataat aaaatgtaat tagttcataa gcaattccca aaacgccgcg tgacccttcg
     3721 tttgcctttg aatacaattt gttttatttt agcgcattta atatatttga gtttcctttt
     3781 tagcgatgac atgataaaac aaaaaacaac aattttagaa aacatttaat taaatgaata
     3841 gaaaaatagg gagattcaac tgaagttact gtgggtggaa atgacaggtc accaggatct
     3901 gaactaagag aaagtctctc tatattttga tatatagaga gagataaaga aggagagagt
     3961 gtgaatatat gtatgtgcat gatactgtga gacttcttca gtaaaggaag cagctttggt
     4021 ttgctgcgga tcgggataaa aacggcgaga agtttgtgat tgttgaagca aaacaacacg
     4081 gaatctgaaa ggaaaggaga agacaaaaaa aaaaaaagat tttaagagga agagagagag
     4141 agatcggttt cgtggctgac ggaaaaaaaa aggggagaga tttagtttta tccaacagtg
     4201 cctgaattac gttctgcaat caatacggac ataaccgtcc gttgtgtcct gtttataaag
     4261 ttttgctttt tttattttct ccattgatgg gtcttttctt tcttctctct ctcgtgtttc
     4321 tttcatgggg ttaagactag tgtttaccgc gtgaaggttt ttttttcttt ctattttctt
     4381 tcatttcctc tccttctact tcttcttctc cagttctcat ctgggttctt caatggtatt
     4441 cacactcgaa gatttcgttg gggactggcg acagacagcc ggctacaacc tggaccaagt
     4501 ccttgaacag ggaggtgtgt ccagtttgtt tcagaatctc ggggtgtccg taactccgat
     4561 ccaaaggatt gtcctgagcg gtgaaaatgg gctgaagatc gacatccatg tcatcatccc
     4621 gtatgaaggt ctgagcggcg accaaatggg ccagatcgaa aaaattttta aggtggtgta
     4681 ccctgtggat gatcatcact ttaaggtgat cctgcactat ggcacactgg taatcgacgg
     4741 ggttacgccg aacatgatcg actatttcgg acggccgtat gaaggcatcg ccgtgttcga
     4801 cggcaaaaag atcactgtaa cagggaccct gtggaacggc aacaaaatta tcgacgagcg
     4861 cctgatcaac cccgacggct ccctgctgtt ccgagtaacc atcaacggag tgaccggctg
     4921 gcggctgtgc gaacgcattc tggcttcgga ttataaggac catgacggag actataagga
     4981 ccatgacctc gacgctgcag cagcggatta taaggacgat gacgataagt gagcttgtca
     5041 agcagatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct
     5101 tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta
     5161 atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta
     5221 atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc
     5281 atctatgtta ctagatcgac gctagcatga agagcaattc tgacaggata tattggcggg
     5341 taaacctaag agaaaagagc gtttattaga ataatcggat atttaaaagg gcgtgaaaag
     5401 gtttatccgt tcgtccattt gtatgtgcat gccaaccaca gggttcccct cgggatcaaa
     5461 gtactttgat ccaacccctc cgctgctata gtgcagtcgg cttctgacgt tcagtgcagc
     5521 cgtcatctga aaacgacatg tcgcacaagt cctaagttac gcgacaggct gccgccctgc
     5581 ccttttcctg gcgttttctt gtcgcgtgtt ttagtcgcat aaagtagaat acttgcgact
     5641 agaaccggag acattacgcc atgaacaaga gcgccgccgc tggcctgctg ggctatgccc
     5701 gcgtcagcac cgacgaccag gacttgacca accaacgggc cgaactgcac gcggccggct
     5761 gcaccaagct gttttccgag aagatcaccg gcaccaggcg cgaccgcccg gagctggcca
     5821 ggatgcttga ccacctacgc cctggcgacg ttgtgacagt gaccaggcta gaccgcctgg
     5881 cccgcagcac ccgcgaccta ctggacattg ccgagcgcat ccaggaggcc ggcgcgggcc
     5941 tgcgtagcct ggcagagccg tgggccgaca ccaccacgcc ggccggccgc atggtgttga
     6001 ccgtgttcgc cggcattgcc gagttcgagc gttccctaat catcgaccgc acccggagcg
     6061 ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc ccgccctacc ctcaccccgg
     6121 cacagatcgc gcacgcccgc gagctgatcg accaggaagg ccgcaccgtg aaagaggcgg
     6181 ctgcactgct tggcgtgcat cgctcgaccc tgtaccgcgc acttgagcgc agcgaggaag
     6241 tgacgcccac cgaggccagg cggcgcggtg ccttccgtga ggacgcattg accgaggccg
     6301 acgccctggc ggccgccgag aatgaacgcc aagaggaaca agcatgaaac cgcaccagga
     6361 cggccaggac gaaccgtttt tcattaccga agagatcgag gcggagatga tcgcggccgg
     6421 gtacgtgttc gagccgcccg cgcacctctc aaccgtgcgg ctgcatgaaa tcctggccgg
     6481 tttgtctgat gccaagctgg cggcctggcc ggccagcttg gccgctgaag aaaccgagcg
     6541 ccgccgtcta aaaaggtgat gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc
     6601 gtatatgatg cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa ggttatcgct
     6661 gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg caacccatct agcccgcgcc
     6721 ctgcaactcg ccggggccga tgttctgtta gtcgattccg atccccaggg cagtgcccgc
     6781 gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga ccgcccgacg
     6841 attgaccgcg acgtgaaggc catcggccgg cgcgacttcg tagtgatcga cggagcgccc
     6901 caggcggcgg acttggctgt gtccgcgatc aaggcagccg acttcgtgct gattccggtg
     6961 cagccaagcc cttacgacat atgggccacc gccgacctgg tggagctggt taagcagcgc
     7021 attgaggtca cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc gatcaaaggc
     7081 acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc cattcttgag
     7141 tcccgtatca cgcagcgcgt gagctaccca ggcactgccg ccgccggcac aaccgttctt
     7201 gaatcagaac ccgagggcga cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa
     7261 tcaaaactca tttgagttaa tgaggtaaag agaaaatgag caaaagcaca aacacgctaa
     7321 gtgccggccg tccgagcgca cgcagcagca aggctgcaac gttggccagc ctggcagaca
     7381 cgccagccat gaagcgggtc aactttcagt tgccggcgga ggatcacacc aagctgaaga
     7441 tgtacgcggt acgccaaggc aagaccatta ccgagctgct atctgaatac atcgcgcagc
     7501 taccagagta aatgagcaaa tgaataaatg agtagatgaa ttttagcggc taaaggaggc
     7561 ggcatggaaa atcaagaaca accaggcacc gacgccgtgg aatgccccat gtgtggagga
     7621 acgggcggtt ggccaggcgt aagcggctgg gttgcctgcc ggccctgcaa tggcactgga
     7681 acccccaagc ccgaggaatc ggcgtgagcg gtcgcaaacc atccggcccg gtacaaatcg
     7741 gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc
     7801 aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc
     7861 gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg
     7921 gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc
     7981 gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg
     8041 tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg
     8101 ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga
     8161 accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg
     8221 acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa
     8281 cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg
     8341 gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga
     8401 gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga
     8461 tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc
     8521 ccggcatcgg c
//
