LOCUS       sequence-482867-        8627 bp    DNA     circular SYN 11-AUG-2025
DEFINITION  Express NanoLuc-YAP1(biased)-TEAD4-HaloTag Fusion Protein in 
            Mammalian Cells under a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8627)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8627)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 11, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: NanoLuc-YAP1(biased)-TEAD4-HaloTag Bidirection...
FEATURES             Location/Qualifiers
     source          1..8627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..546
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     447..466
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     663..680
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    830..878
                     /note="synthetic polyadenylation signal"
     CDS             complement(902..1300)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      1297..1306
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1331..1688)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1345..1480
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        1497..1688
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     1645..1665
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    1767..1848
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(3503..4015)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     enhancer        4078..4381
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4382..4585
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4536..4556
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          4721..4853
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4836..4855
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     4897..4916
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4897..4915
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6252..6272
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             6288..7172
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     polyA_signal    7259..7380
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7270..7289)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(7495..7528)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        7538..7585
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7617..8411
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7671..7690)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     8281..8300
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa ccgccttgca
       61 gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta actggcttgg
      121 aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taaccggcgc atgacttcaa
      181 gactaactcc tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt gcatgtcttt
      241 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcggac tgaacggggg
      301 gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc ggaactgagt gtcaggcgtg
      361 gaatgagaca aacgcggcca taacagcgga atgacaccgg taaaccgaaa ggcaggaaca
      421 ggagagcgca cgagggagcc gccaggggga aacgcctggt atctttatag tcctgtcggg
      481 tttcgccacc actgatttga gcgtcagatt tcgtgatgct tgtcaggggg gcggagccta
      541 tggaaaaacg gctttgccgc ggccctctca cttccctgtt aagtatcttc ctggcatctt
      601 ccaggaaatc tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga acgaccgagc
      661 gtagcgagtc agtgagcgag gaagcggaat atatcctgta tcacatattc tgctgacgca
      721 ccggtgcagc cttttttctc ctgccacatg aagcacttca ctgacaccct catcagtgcc
      781 aacatagtaa gccagtatac actccgctag cgctgatgtc cggaatcgtc acacaaaaaa
      841 ccaacacaca gatgtaatga aaataaagat attttattgc ggccatcgtg atggctagag
      901 tttagccctc ccacacataa ccagagggca gcaattcacg aatcccaact gccgtcggct
      961 gtccatcact gtccttcact atggctttga tcccaggatg cagatcgaga agcacctgtc
     1021 ggcaccgtcc gcaggggctc aagatgcccc tgttctcatt tccgatcgcg acgatacaag
     1081 tcaggttgcc agctgccgca gcagcagcag tgcccagcac cacgagttct gcacaaggtc
     1141 ccccagtaaa atgatataca ttgacaccag tgaagatgcg gccgtcgcta gagagagctg
     1201 cgctggcgac gctgtagtct tcagagatgg ggatgctgtt gattgtagcc gttgctcttt
     1261 caatgagggt ggattcttct tgagacaaag gcttggccat ggtggcgcta gtgtcagaag
     1321 aatcgagctt tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata
     1381 gctcagaggc cgaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg
     1441 gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga
     1501 ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     1561 gggactttcc acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg
     1621 ctggggagcc tggggacttt ccacacccta actgacacac attccacagc tggttctttc
     1681 cgcctcagaa ggtacctaac caagttcctc tttcagaggt tatttcaggc catggtgctg
     1741 cgcaagacgc gttaattaag acgtcggcag tgaaaaaaat gctttatttg tgaaatttgt
     1801 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     1861 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     1921 aacctctaca aatgtggtat ggctgattat gatccgcggc cgcttaaact aaccatgtaa
     1981 gaaagctttc tttatctagc ttggtggcag ccaaaacaga ctccatgtca ttaaggatgt
     2041 cagaactcaa agcttcctgc agacttggca tcagctcctc tccttctatg ttcattccat
     2101 ctccttccag tgttccaagg tccacatttg tcccaggaat ggcttcaagg tagtctggga
     2161 aacggttctg ctgtgagggc agggtgcttt ggttgatagt atcacctgta tccatctcat
     2221 ccacactgtt caggaagtca tctggggttc gagggacact gtagctgctc atgcttagtc
     2281 cactgtctgt actctcatct cgagagtgat aggtgccact gttaaggaaa ggatctgagc
     2341 tattggtcgt cattgttctc aattcctgag acatcccggg agaagacact ggattttgag
     2401 tcccaccatc ctgctccagt gttggtaact ggctacgcag ggctaactcc tgacattttg
     2461 gagaatttgc tgtgctggga ttgatattcc gcattgcctg ccgaagcagt tcttgctgtt
     2521 tcagccgcag cctctccttc tccatctgca gttgctgcag tcgcatctgt tgctgctggt
     2581 tggagttgct gccacccatg acgcctccct gtgggctctg gggagccagg ggtggtggct
     2641 gtttcactgg agcactctga ctgattctct ggttcatggc aaaacgaggg tcaagccttg
     2701 ggtctagcca agaggtggtc ttgttcttat ggtttatata gtaaatttct ccatcctgag
     2761 tcatggcttg ttcccatcca tcaggaagag gacctgaagc cgagttcatc atattctgct
     2821 gcactggtgg actggtgggg gctgtgacgt tcatctggga cagcatggcc ttcctggggt
     2881 cctgccatgt tgttgtctga tcgatgtgat ttaagaagta tctctgacca gaagatgtct
     2941 ttgccatctc ccaacctgct ggcagaggta catcatcagg tatctcaaaa gaagactgtc
     3001 gaagatgctg agctgtgggt gtagctgctg ggccagagac tactccagtg ggggtcagtg
     3061 tcccaggaga aacagctccc aactgcagag aagctggaga ggaatgagct cgaacatgct
     3121 gtggagtcag ggctcctgca gtgcctgcat cagtactggc ctgtcgggag tgggatttgg
     3181 gctccggcgg cttgaagaag gagtcgggca gcttccggag cctcatgggc acggtctggg
     3241 gcacgttggc cgtcttgggg ttcatgacgg cgttgaagag cgcctccagg tcggtctccg
     3301 agtccccgcg gacgtgcacg atctgatgcc cggcgggggg tgcctgcggc gccgcctggg
     3361 tcgccgcggg tgccggttgc ccgggtccgg acggcgggcc ctgcccctgc gggggctgcg
     3421 aaggcggctg cccttggccc tggggggccg gttgaggcgg cggctgctgc ccgggatcca
     3481 tggcgatcgc gccgctcgag cccgccagaa tgcgttcgca cagccgccag ccggtcactc
     3541 cgttgatggt tactcggaac agcagggagc cgtcggggtt gatcaggcgc tcgtcgataa
     3601 ttttgttgcc gttccacagg gtccctgtta cagtgatctt tttgccgtcg aacacggcga
     3661 tgccttcata cggccgtccg aaatagtcga tcatgttcgg cgtaaccccg tcgattacca
     3721 gtgtgccata gtgcaggatc accttaaagt gatgatcatc cacagggtac accaccttaa
     3781 aaattttttc gatctggccc atttggtcgc cgctcagacc ttcatacggg atgatgacat
     3841 ggatgtcgat cttcagccca ttttcaccgc tcaggacaat cctttggatc ggagttacgg
     3901 acaccccgag attctgaaac aaactggaca cacctccctg ttcaaggact tggtccaggt
     3961 tgtagccggc tgtctgtcgc cagtccccaa cgaaatcttc gagtgtgaag accatactag
     4021 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     4081 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     4141 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     4201 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     4261 tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     4321 gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     4381 ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt
     4441 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     4501 ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg
     4561 gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatcacta gaagctttat
     4621 tgcggtagtt tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc
     4681 gaacttaagc tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca aggttacaag
     4741 acaggtttaa ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt
     4801 ctgataggca cctattggtc ttactgacat ccactttgcc tttctctcca caggtgtcca
     4861 ctcccagttc aattacagct cttaaggcta gagtattaat acgactcact atagggctag
     4921 cgatcgccat ggagggcacg gccggcacca ttacctccaa cgagtggagc tctcccacct
     4981 cccctgaggg gagcaccgcc tctgggggca gtcaggcact ggacaagccc atcgacaatg
     5041 acgcagaggg cgtgtggagc ccggatattg agcagagttt ccaggaggcc ctcgccatct
     5101 acccgccctg tggcaggcgc aaaatcatcc tgtcggacga gggcaagatg tatggtcgga
     5161 acgagctgat tgcccgctac atcaagctcc ggacagggaa gacccgcacc aggaagcagg
     5221 tctccagcca catccaggtg ctggctcgtc gcaaagctcg cgagatccag gccaagctaa
     5281 aggaccaggc agctaaggac aaggccctgc agagcatggc tgccatgtcg tctgcacaga
     5341 tcatctccgc cacggccttc cacagtagca tggccctcgc ccggggcccc ggccgcccag
     5401 cagtctcagg gttttggcaa ggagctttgc caggccaagc cggaacgtcc catgatgtga
     5461 agcctttctc tcagcaaacc tatgctgtcc agcctccgct gcctctgcca gggtttgagt
     5521 ctcctgcagg gcccgcccca tcgccctctg cgcccccggc acccccatgg cagggccgca
     5581 gcgtggccag ctccaagctc tggatgttgg agttctctgc cttcctggag cagcagcagg
     5641 acccggacac gtacaacaag cacctgttcg tgcacattgg ccagtccagc ccaagctaca
     5701 gcgaccccta cctcgaagcc gtggacatcc gccaaatcta tgacaaattc ccggagaaaa
     5761 agggtggact caaggatctc ttcgaacggg gaccctccaa tgcctttttt cttgtgaagt
     5821 tctgggcaga cctcaacacc aacatcgagg atgaaggcag ctccttctat ggggtctcca
     5881 gccagtatga gagccccgag aacatgatca tcacctgctc cacgaaggtc tgctctttcg
     5941 gcaagcaggt ggtggagaaa gttgagacag agtatgctcg ctatgagaat ggacactact
     6001 cttaccgcat ccaccggtcc ccgctctgtg agtacatgat caacttcatc cacaagctca
     6061 agcacctccc tgagaagtac atgatgaaca gcgtgctgga gaacttcacc atcctgcagg
     6121 tggtcaccaa cagagacaca caggagacct tgctgtgcat tgcctatgtc tttgaggtgt
     6181 cagccagtga gcacggggct cagcaccaca tctacaggct ggtgaaagaa gtttctctcg
     6241 agccaaccac tgaggatctg tactttcaga gcgataacga tggatccgaa atcggtactg
     6301 gctttccatt cgacccccat tatgtggaag tcctgggcga gcgcatgcac tacgtcgatg
     6361 ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg taacccgacc tcctcctacg
     6421 tgtggcgcaa catcatcccg catgttgcac cgacccatcg ctgcattgct ccagacctga
     6481 tcggtatggg caaatccgac aaaccagacc tgggttattt cttcgacgac cacgtccgct
     6541 tcatggatgc cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc attcacgact
     6601 ggggctccgc tctgggtttc cactgggcca agcgcaatcc agagcgcgtc aaaggtattg
     6661 catttatgga gttcatccgc cctatcccga cctgggacga atggccagaa tttgcccgcg
     6721 agaccttcca ggccttccgc accaccgacg tcggccgcaa gctgatcatc gatcagaacg
     6781 tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa gtcgagatgg
     6841 accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg cgcttcccaa
     6901 acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa gaatacatgg
     6961 actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca ggcgttctga
     7021 tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag gctgtggaca
     7081 tcggcccggg tctgaatctg ctgcaagaag acaacccgga cctgatcggc agcgagatcg
     7141 cgcgctggct gtctactctg gagatttccg gttaatagaa ttctagagtc gacctgcagg
     7201 catgcaagct gatttggctg ctgccaccgc tgagcaaggc cgcttcgagc agacatgata
     7261 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     7321 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     7381 aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg ggaggttttt
     7441 ttaagcaagt aaaacctcta caaatgtggt aaaatcttaa ttaagaattc taatataact
     7501 tcgtatagca tacattatac gaagttatgg atccactgtt gacaattaat catcggcata
     7561 gtatatcggc atagtataat acgacaaggt gaggaactaa acccaggagg cagatcatga
     7621 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     7681 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     7741 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     7801 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     7861 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     7921 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     7981 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     8041 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     8101 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     8161 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     8221 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     8281 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     8341 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     8401 agttcttctg attcgaaatg accgaccaag cgacgcccaa ccggtatcag ctcactcaaa
     8461 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtcaaacat
     8521 gagaattaca acttatatcg tatggggctg acttcaggtg ctacatttga agagataaat
     8581 tgcactgaaa tctagaaata ttttatctga ttaataagat gatcttc
//
