LOCUS       sequence-386842-        8325 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MoClo Level 1 luciferase reporter: proANAC032::LucF::flag::notT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8325)
  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 8325)
  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: pEPSW1KN0014
FEATURES             Location/Qualifiers
     source          1..8325
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      314..508
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(744..766)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    852..992
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     907..926
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1007..1024)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1178..1766)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1258..1277)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1854..2669)
                     /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(1854..2669)
                     /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     2577..2596
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    3102..3126
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             4229..4741
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             4802..4825
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      4841..5093
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_feature    5117..5141
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             6442..7071
                     /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(7500..8325,1..248)
                     /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 gccgttttct ctaccgcctg gcacgccgcg ccgcaggcaa ggcagaagcc agatggttgt
       61 tcaagacgat ctacgaacgc agtggcagcg ccggagagtt caagaagttc tgtttcaccg
      121 tgcgcaagct gatcgggtca aatgacctgc cggagtacga tttgaaggag gaggcggggc
      181 aggctggccc gatcctagtc atgcgctacc gcaacctgat cgagggcgaa gcatccgccg
      241 gttcctaatg tacggagcag atgctagggc aaattgccct agcaggggaa aaaggtcgaa
      301 aaggactctt tcctgtggat agcacgtaca ttgggaaccc aaagccgtac attgggaacc
      361 ggaacccgta cattgggaac ccaaagccgt acattgggaa ccggtcacac atgtaagtga
      421 ctgatataaa agagaaaaaa ggcgattttt ccgcctaaaa ctctttaaaa cttattaaaa
      481 ctcttaaaac ccgcctggcc tgtgcataac tgtctggcca gcgcacagcc gaagtgctgc
      541 aaaaagcgcc tacccttcgg tcgctgcgct ccctacgccc cgccgcttcg cgtcggccta
      601 tcgcggccgc tggccgctca aaaatggctg gcctacggcc aggcaatcta ccagggcgcg
      661 gacaagccgc gccgtcgcca ctcgaccgcc ggcgcccaca tcaaggcacc ctgcctcgcg
      721 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggtgac ggtcacagct
      781 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
      841 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
      901 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
      961 acagatgcgt aaggagaaaa taccgcatca ggcgctcatc cgcttcctcg ctcactgact
     1021 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     1081 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     1141 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     1201 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     1261 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     1321 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     1381 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     1441 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     1501 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     1561 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     1621 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     1681 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     1741 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     1801 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gcattctagg tgattagaaa
     1861 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     1921 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     1981 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     2041 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     2101 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     2161 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     2221 gcgagtcgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     2281 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     2341 aatacctgga atgctgtttt ccctgggatc gcagtggtga gtaaccatgc atcatcagga
     2401 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     2461 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     2521 ggcgcatcgg gcttcccata caatcggtag attgtcgcac ctgattgccc gacattatcg
     2581 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggccttgag
     2641 caagacgttt cccgttgaat atggctcata acagaactta ttatttcctt cctcttttct
     2701 acagtattta aagatacccc aagaagctaa ttataacaag acgaactcca attcactgtt
     2761 ccttgcattc taaaacctta aataccagaa aacagctttt tcaaagttgt tttcaaagtt
     2821 ggcgtataac atagtatcga cggagccgat tttgaaaccg cggtgatcac aggcagcaac
     2881 gctctgtcat cgttacaatc aacatgctac cctccgcgag atcatccgtg tttcaaaccc
     2941 ggcagcttag ttgccgttct tccgaatagc atcggtaaca tgagcaaagt ctgccgcctt
     3001 acaacggctc tcccgctgac gccgtcccgg actgatgggc tgcctgtatc gagtggtgat
     3061 tttgtgccga gctgccggtc ggggagctgt tggctggctg gtggcaggat atattgtggt
     3121 gtaaacataa cgaattgctc ttcaatgagg agttgagctt tttatcaaat tcaacccgcc
     3181 acaataaaaa gcggcagaac taacattccg gctccacgtg gcatagatcg aagcgtttat
     3241 ctattaggtg gcacaggcgg ctcgagatga cgttcttgtg gactccatta gagaaaatga
     3301 cagaattaag tgggcattag cataagttga taactatgag gtgcttttta aaaagaaaac
     3361 taacagctgc gattctgttt ttttttgccg ttagtgtacg ctaaatcata gacctcttcg
     3421 tggttataaa gaaagaaact taatttgtct cgaatttgag tttggatgcg acgttttacg
     3481 tactagtttt ataaaagtaa atgttataga ccttgaattt caaagtggaa tgtattttac
     3541 acacacaatt gatacaccaa caattgtgga agcaaggata tagagtatcg atctttaatg
     3601 gagaagaata tatccttaaa gtaatcatta ctttttaatt acggcggaaa gagaatagtt
     3661 aaatttacgg cgaaaagaat gttcattaat caattgatga cgttcgttta taatttcaaa
     3721 taagatcgaa aataacaggt gaacaaagaa aaaatagaca ttaaaaatag taagatcgaa
     3781 aatacaaccc ttaaaaatct cacgaaatca atgagagaaa caattttgag atacggtcaa
     3841 cgtttggtca cctttaggaa agtacacgca taagatcgaa cgtgtcgatt cttattgctc
     3901 gttcccaaaa tcgacatgct ctcggaccgc tacattccaa atagtctgac gtaagcaatg
     3961 acaaaactca cctacattac agtctgccac gtgtccctct ccatctactt caaatgaatc
     4021 ctccactata cagatttaac catttttcta tttttgtttg agattccttt ccaccaacac
     4081 aaagcccctg tctatccgcc atgtgtccac gacctttctt atatatatag tcatctttct
     4141 tctttgctcg agagaagttt caactttcaa acatatcttt acagttcttt cttgctaaac
     4201 aacaataaag gggaaaatag gttaattaat ggtattcaca ctcgaagatt tcgttgggga
     4261 ctggcgacag acagccggct acaacctgga ccaagtcctt gaacagggag gtgtgtccag
     4321 tttgtttcag aatctcgggg tgtccgtaac tccgatccaa aggattgtcc tgagcggtga
     4381 aaatgggctg aagatcgaca tccatgtcat catcccgtat gaaggtctga gcggcgacca
     4441 aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc atcactttaa
     4501 ggtgatcctg cactatggca cactggtaat cgacggggtt acgccgaaca tgatcgacta
     4561 tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca ctgtaacagg
     4621 gaccctgtgg aacggcaaca aaattatcga cgagcgcctg atcaaccccg acggctccct
     4681 gctgttccga gtaaccatca acggagtgac cggctggcgg ctgtgcgaac gcattctggc
     4741 ttcggattat aaggaccatg acggagacta taaggaccat gacctcgacg ctgcagcagc
     4801 ggattataag gacgatgacg ataagtgagc ttgtcaagca gatcgttcaa acatttggca
     4861 ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct
     4921 gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg
     4981 ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata
     5041 gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgacgcta
     5101 gcatgaagag caattctgac aggatatatt ggcgggtaaa cctaagagaa aagagcgttt
     5161 attagaataa tcggatattt aaaagggcgt gaaaaggttt atccgttcgt ccatttgtat
     5221 gtgcatgcca accacagggt tcccctcggg atcaaagtac tttgatccaa cccctccgct
     5281 gctatagtgc agtcggcttc tgacgttcag tgcagccgtc atctgaaaac gacatgtcgc
     5341 acaagtccta agttacgcga caggctgccg ccctgccctt ttcctggcgt tttcttgtcg
     5401 cgtgttttag tcgcataaag tagaatactt gcgactagaa ccggagacat tacgccatga
     5461 acaagagcgc cgccgctggc ctgctgggct atgcccgcgt cagcaccgac gaccaggact
     5521 tgaccaacca acgggccgaa ctgcacgcgg ccggctgcac caagctgttt tccgagaaga
     5581 tcaccggcac caggcgcgac cgcccggagc tggccaggat gcttgaccac ctacgccctg
     5641 gcgacgttgt gacagtgacc aggctagacc gcctggcccg cagcacccgc gacctactgg
     5701 acattgccga gcgcatccag gaggccggcg cgggcctgcg tagcctggca gagccgtggg
     5761 ccgacaccac cacgccggcc ggccgcatgg tgttgaccgt gttcgccggc attgccgagt
     5821 tcgagcgttc cctaatcatc gaccgcaccc ggagcgggcg cgaggccgcc aaggcccgag
     5881 gcgtgaagtt tggcccccgc cctaccctca ccccggcaca gatcgcgcac gcccgcgagc
     5941 tgatcgacca ggaaggccgc accgtgaaag aggcggctgc actgcttggc gtgcatcgct
     6001 cgaccctgta ccgcgcactt gagcgcagcg aggaagtgac gcccaccgag gccaggcggc
     6061 gcggtgcctt ccgtgaggac gcattgaccg aggccgacgc cctggcggcc gccgagaatg
     6121 aacgccaaga ggaacaagca tgaaaccgca ccaggacggc caggacgaac cgtttttcat
     6181 taccgaagag atcgaggcgg agatgatcgc ggccgggtac gtgttcgagc cgcccgcgca
     6241 cctctcaacc gtgcggctgc atgaaatcct ggccggtttg tctgatgcca agctggcggc
     6301 ctggccggcc agcttggccg ctgaagaaac cgagcgccgc cgtctaaaaa ggtgatgtgt
     6361 atttgagtaa aacagcttgc gtcatgcggt cgctgcgtat atgatgcgat gagtaaataa
     6421 acaaatacgc aaggggaacg catgaaggtt atcgctgtac ttaaccagaa aggcgggtca
     6481 ggcaagacga ccatcgcaac ccatctagcc cgcgccctgc aactcgccgg ggccgatgtt
     6541 ctgttagtcg attccgatcc ccagggcagt gcccgcgatt gggcggccgt gcgggaagat
     6601 caaccgctaa ccgttgtcgg catcgaccgc ccgacgattg accgcgacgt gaaggccatc
     6661 ggccggcgcg acttcgtagt gatcgacgga gcgccccagg cggcggactt ggctgtgtcc
     6721 gcgatcaagg cagccgactt cgtgctgatt ccggtgcagc caagccctta cgacatatgg
     6781 gccaccgccg acctggtgga gctggttaag cagcgcattg aggtcacgga tggaaggcta
     6841 caagcggcct ttgtcgtgtc gcgggcgatc aaaggcacgc gcatcggcgg tgaggttgcc
     6901 gaggcgctgg ccgggtacga gctgcccatt cttgagtccc gtatcacgca gcgcgtgagc
     6961 tacccaggca ctgccgccgc cggcacaacc gttcttgaat cagaacccga gggcgacgct
     7021 gcccgcgagg tccaggcgct ggccgctgaa attaaatcaa aactcatttg agttaatgag
     7081 gtaaagagaa aatgagcaaa agcacaaaca cgctaagtgc cggccgtccg agcgcacgca
     7141 gcagcaaggc tgcaacgttg gccagcctgg cagacacgcc agccatgaag cgggtcaact
     7201 ttcagttgcc ggcggaggat cacaccaagc tgaagatgta cgcggtacgc caaggcaaga
     7261 ccattaccga gctgctatct gaatacatcg cgcagctacc agagtaaatg agcaaatgaa
     7321 taaatgagta gatgaatttt agcggctaaa ggaggcggca tggaaaatca agaacaacca
     7381 ggcaccgacg ccgtggaatg ccccatgtgt ggaggaacgg gcggttggcc aggcgtaagc
     7441 ggctgggttg cctgccggcc ctgcaatggc actggaaccc ccaagcccga ggaatcggcg
     7501 tgagcggtcg caaaccatcc ggcccggtac aaatcggcgc ggcgctgggt gatgacctgg
     7561 tggagaagtt gaaggccgcg caggccgccc agcggcaacg catcgaggca gaagcacgcc
     7621 ccggtgaatc gtggcaagcg gccgctgatc gaatccgcaa agaatcccgg caaccgccgg
     7681 cagccggtgc gccgtcgatt aggaagccgc ccaagggcga cgagcaacca gattttttcg
     7741 ttccgatgct ctatgacgtg ggcacccgcg atagtcgcag catcatggac gtggccgttt
     7801 tccgtctgtc gaagcgtgac cgacgagctg gcgaggtgat ccgctacgag cttccagacg
     7861 ggcacgtaga ggtttccgca gggccggccg gcatggccag tgtgtgggat tacgacctgg
     7921 tactgatggc ggtttcccat ctaaccgaat ccatgaaccg ataccgggaa gggaagggag
     7981 acaagcccgg ccgcgtgttc cgtccacacg ttgcggacgt actcaagttc tgccggcgag
     8041 ccgatggcgg aaagcagaaa gacgacctgg tagaaacctg cattcggtta aacaccacgc
     8101 acgttgccat gcagcgtacg aagaaggcca agaacggccg cctggtgacg gtatccgagg
     8161 gtgaagcctt gattagccgc tacaagatcg taaagagcga aaccgggcgg ccggagtaca
     8221 tcgagatcga gctagctgat tggatgtacc gcgagatcac agaaggcaag aacccggacg
     8281 tgctgacggt tcaccccgat tactttttga tcgatcccgg catcg
//
