LOCUS       sequence-475176-        6510 bp    DNA     circular SYN 07-JUL-2025
DEFINITION  Express PPP2R1A-NanoLuc 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 6510)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 6510)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: NanoBRET Assay Vector, PPP2R1A-NanoLuc
FEATURES             Location/Qualifiers
     source          1..6510
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1033..1052
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1033..1051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1063..1072
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2853..3365
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(3453..3471)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3508..3629
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3519..3538)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3573..3592
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3874..4231
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3874..4065
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3897..3917)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4082..4217
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4246..4293
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4325..5119
                     /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(4379..4398)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4989..5008
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5183..5231
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(5470..6058)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5550..5569)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6174..6457
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcgat cgccatggcg gcggccgacg
     1081 gcgacgactc gctgtacccc atcgcggtgc tcatagacga actccgcaat gaggacgttc
     1141 agcttcgcct caacagcatc aagaagctgt ccaccatcgc cttggccctt ggggttgaaa
     1201 ggacccgaag tgagcttctg cctttcctta cagataccat ctatgatgaa gatgaggtcc
     1261 tcctggccct ggcagaacag ctgggaacct tcactaccct ggtgggaggc ccagagtacg
     1321 tgcactgcct gctgccaccg ctggagtcgc tggccacagt ggaggagaca gtggtgcggg
     1381 acaaggcagt ggagtcctta cgggccatct cacacgagca ctcgccctct gacctggagg
     1441 cgcactttgt gccgctagtg aagcggctgg cgggcggcga ctggttcacc tcccgcacct
     1501 cggcctgcgg cctcttctcc gtctgctacc cccgagtgtc cagtgctgtg aaggcggaac
     1561 ttcgacagta cttccggaac ctgtgctcag atgacacccc catggtgcgg cgggccgcag
     1621 cctccaagct gggggagttt gccaaggtgc tggagctgga caacgtcaag agtgagatca
     1681 tccccatgtt ctccaacctg gcctctgacg agcaggactc ggtgcggctg ctggcggtgg
     1741 aggcgtgcgt gaacatcgcc cagcttctgc cccaggagga tctggaggcc ctggtgatgc
     1801 ccactctgcg ccaggccgct gaagacaagt cctggcgcgt ccgctacatg gtggctgaca
     1861 agttcacaga gctccagaaa gcagtggggc ctgagatcac caagacagac ctggtccctg
     1921 ccttccagaa cctgatgaaa gactgtgagg ccgaggtgag ggccgcagcc tcccacaagg
     1981 tcaaagagtt ctgtgaaaac ctctcagctg actgtcggga gaatgtgatc atgtcccaga
     2041 tcttgccctg catcaaggag ctggtgtccg atgccaacca acatgtcaag tctgccctgg
     2101 cctcagtcat catgggtctc tctcccatct tgggcaaaga caacaccatc gagcacctct
     2161 tgcccctctt cctggctcag ctgaaggatg agtgccctga ggtacggctg aacatcatct
     2221 ctaacctgga ctgtgtgaac gaggtgattg gcatccggca gctgtcccag tccctgctcc
     2281 ctgccattgt ggagctggct gaggacgcca agtggcgggt gcggctggcc atcattgagt
     2341 acatgcccct cctggctgga cagctgggag tggagttctt tgatgagaaa cttaactcct
     2401 tgtgcatggc ctggcttgtg gatcatgtat atgccatccg cgaggcagcc accagcaacc
     2461 tgaagaagct agtggaaaag tttgggaagg agtgggccca tgccacaatc atccccaagg
     2521 tcttggccat gtccggagac cccaactacc tgcaccgcat gactacgctc ttctgcatca
     2581 atgtgctgtc tgaggtctgt gggcaggaca tcaccaccaa gcacatgcta cccacggttc
     2641 tgcgcatggc tggggacccg gttgccaatg tccgcttcaa tgtggccaag tctctgcaga
     2701 agatagggcc catcctggac aacagcacct tgcagagtga agtcaagccc atcctagaga
     2761 agctgaccca ggaccaggat gtggacgtca aatactttgc ccaggaggct ctgactgttc
     2821 tgtctctcgc cgtttctctc ggctcgagcg gcgtcttcac actcgaagat ttcgttgggg
     2881 actggcgaca gacagccggc tacaacctgg accaagtcct tgaacaggga ggtgtgtcca
     2941 gtttgtttca gaatctcggg gtgtccgtaa ctccgatcca aaggattgtc ctgagcggtg
     3001 aaaatgggct gaagatcgac atccatgtca tcatcccgta tgaaggtctg agcggcgacc
     3061 aaatgggcca gatcgaaaaa atttttaagg tggtgtaccc tgtggatgat catcacttta
     3121 aggtgatcct gcactatggc acactggtaa tcgacggggt tacgccgaac atgatcgact
     3181 atttcggacg gccgtatgaa ggcatcgccg tgttcgacgg caaaaagatc actgtaacag
     3241 ggaccctgtg gaacggcaac aaaattatcg acgagcgcct gatcaacccc gacggctccc
     3301 tgctgttccg agtaaccatc aacggagtga ccggctggcg gctgtgcgaa cgcattctgg
     3361 cgtaaggccg cgactctaga gtcgacctgc aggcatgcaa gctgatccgg ctgctaacaa
     3421 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     3481 tggggcggcc gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa
     3541 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     3601 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     3661 aggttcaggg ggagatgtgg gaggtttttt taagcaagta aaacctctac aaatgtggta
     3721 aaatcgaatt ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta
     3781 cgcatctgtg cggtatttca caccgcatac gcggatctgc gcagcaccat ggcctgaaat
     3841 aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa
     3901 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3961 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     4021 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     4081 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4141 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     4201 aggctttttt ggaggcctag gcttttgcaa aaagcttaat taactgttga caattaatca
     4261 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac ccaggaggca
     4321 gatcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     4381 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     4441 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     4501 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     4561 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     4621 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     4681 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     4741 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     4801 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     4861 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     4921 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     4981 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     5041 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     5101 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     5161 aacctgccat cacgatggcc gcaataaaat atctttattt tcattacatc tgtgtgttgg
     5221 ttttttgtgt gaatcgatag cgataaggat cctctttgcg cttgcgtttt cccttgtcca
     5281 gatagcccag tagctgacat tcatccgggg tcagcaccgt ttctgcggac tggctttcta
     5341 cccggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5401 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5461 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5521 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5581 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5641 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5701 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5761 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5821 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5881 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     5941 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6001 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatttcaaga
     6061 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     6121 gattttggtc atgagattat caaaaaggat cttcacctag atccttttat agtccggaaa
     6181 tacaggaacg cacgctggat ggcccttcgc tgggatggtg aaaccatgaa aaatggcagc
     6241 ttcagtggat taagtggggg taatgtggcc tgtaccctct ggttgcatag gtattcatac
     6301 ggttaaaatt tatcaggcgc gattgcggca gtttttcggg tggtttgttg ccatttttac
     6361 ctgtctgctg ccgtgatcgc gctgaacgcg ttttagcggt gcgtacaatt aagggattat
     6421 ggtaaatcca cttactgtct gccctcgtag ccatcgagat aaaccgcagt actccggcca
     6481 cgatgcgtcc ggcgtagagg atcgagatct
//
