LOCUS       sequence-486822-        6536 bp    DNA     circular SYN 22-SEP-2025
DEFINITION  Express NanoLuc-PPP2R1A 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 6536)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6536)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 22, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: NanoBRET Assay Vector, NanoLuc-PPP2R1A
FEATURES             Location/Qualifiers
     source          1..6536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(668..686)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    723..844
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(734..753)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     788..807
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        1089..1446
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1089..1280
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1112..1132)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1297..1432
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        1461..1508
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1540..2334
                     /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(1594..1613)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2204..2223
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2398..2446
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(2685..3273)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2765..2784)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    3389..3672
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     enhancer        3863..4242
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4243..4446
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4397..4417
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          4582..4714
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4697..4716
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     4758..4777
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4758..4776
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4790..5302
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     regulatory      5322..5331
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 tccctgccat tgtggagctg gctgaggacg ccaagtggcg ggtgcggctg gccatcattg
       61 agtacatgcc cctcctggct ggacagctgg gagtggagtt ctttgatgag aaacttaact
      121 ccttgtgcat ggcctggctt gtggatcatg tatatgccat ccgcgaggca gccaccagca
      181 acctgaagaa gctagtggaa aagtttggga aggagtgggc ccatgccaca atcatcccca
      241 aggtcttggc catgtccgga gaccccaact acctgcaccg catgactacg ctcttctgca
      301 tcaatgtgct gtctgaggtc tgtgggcagg acatcaccac caagcacatg ctacccacgg
      361 ttctgcgcat ggctggggac ccggttgcca atgtccgctt caatgtggcc aagtctctgc
      421 agaagatagg gcccatcctg gacaacagca ccttgcagag tgaagtcaag cccatcctag
      481 agaagctgac ccaggaccag gatgtggacg tcaaatactt tgcccaggag gctctgactg
      541 ttctgtctct cgccgtttaa acgaattcgg gctcggtacc cggggatcct ctagagtcga
      601 cctgcaggca tgcaagctga tccggctgct aacaaagccc gaaaggaagc tgagttggct
      661 gctgccaccg ctgagcaata actagcataa ccccttgggg cggccgcttc gagcagacat
      721 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
      781 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
      841 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt
      901 ttttttaagc aagtaaaacc tctacaaatg tggtaaaatc gaattttaac aaaatattaa
      961 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     1021 catacgcgga tctgcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta
     1081 ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag
     1141 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     1201 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     1261 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     1321 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     1381 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     1441 tgcaaaaagc ttaattaact gttgacaatt aatcatcggc atagtatatc ggcatagtat
     1501 aatacgacaa ggtgaggaac taaacccagg aggcagatca tgattgaaca agatggattg
     1561 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     1621 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     1681 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     1741 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     1801 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     1861 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     1921 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     1981 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     2041 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     2101 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     2161 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     2221 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     2281 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     2341 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga tggccgcaat
     2401 aaaatatctt tattttcatt acatctgtgt gttggttttt tgtgtgaatc gatagcgata
     2461 aggatcctct ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc
     2521 cggggtcagc accgtttctg cggactggct ttctacccgg tatcagctca ctcaaaggcg
     2581 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2641 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2701 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2761 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2821 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2881 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2941 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3001 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3061 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3121 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3181 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3241 cagcagatta cgcgcagaaa aaaaggattt caagaagatc ctttgatctt ttctacgggg
     3301 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3361 aggatcttca cctagatcct tttatagtcc ggaaatacag gaacgcacgc tggatggccc
     3421 ttcgctggga tggtgaaacc atgaaaaatg gcagcttcag tggattaagt gggggtaatg
     3481 tggcctgtac cctctggttg cataggtatt catacggtta aaatttatca ggcgcgattg
     3541 cggcagtttt tcgggtggtt tgttgccatt tttacctgtc tgctgccgtg atcgcgctga
     3601 acgcgtttta gcggtgcgta caattaaggg attatggtaa atccacttac tgtctgccct
     3661 cgtagccatc gagataaacc gcagtactcc ggccacgatg cgtccggcgt agaggatcga
     3721 gatcttcaat attggccatt agccatatta ttcattggtt atatagcata aatcaatatt
     3781 ggctattggc cattgcatac gttgtatcta tatcataata tgtacattta tattggctca
     3841 tgtccaatat gaccgccatg ttggcattga ttattgacta gttattaata gtaatcaatt
     3901 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     3961 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     4021 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     4081 actgcccact tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc
     4141 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct
     4201 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     4261 tacaccaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     4321 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaat
     4381 aaccccgccc cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     4441 agagctggtt tagtgaaccg tcagatcact agaagcttta ttgcggtagt ttatcacagt
     4501 taaattgcta acgcagtcag tgcttctgac acaacagtct cgaacttaag ctgcagaagt
     4561 tggtcgtgag gcactgggca ggtaagtatc aaggttacaa gacaggttta aggagaccaa
     4621 tagaaactgg gcttgtcgag acagagaaga ctcttgcgtt tctgataggc acctattggt
     4681 cttactgaca tccactttgc ctttctctcc acaggtgtcc actcccagtt caattacagc
     4741 tcttaaggct agagtattaa tacgactcac tatagggcta gcgctcacca tggtcttcac
     4801 actcgaagat ttcgttgggg actggcgaca gacagccggc tacaacctgg accaagtcct
     4861 tgaacaggga ggtgtgtcca gtttgtttca gaatctcggg gtgtccgtaa ctccgatcca
     4921 aaggattgtc ctgagcggtg aaaatgggct gaagatcgac atccatgtca tcatcccgta
     4981 tgaaggtctg agcggcgacc aaatgggcca gatcgaaaaa atttttaagg tggtgtaccc
     5041 tgtggatgat catcacttta aggtgatcct gcactatggc acactggtaa tcgacggggt
     5101 tacgccgaac atgatcgact atttcggacg gccgtatgaa ggcatcgccg tgttcgacgg
     5161 caaaaagatc actgtaacag ggaccctgtg gaacggcaac aaaattatcg acgagcgcct
     5221 gatcaacccc gacggctccc tgctgttccg agtaaccatc aacggagtga ccggctggcg
     5281 gctgtgcgaa cgcattctgg cgggctcgag cggcgcgatc gccatggcgg cggccgacgg
     5341 cgacgactcg ctgtacccca tcgcggtgct catagacgaa ctccgcaatg aggacgttca
     5401 gcttcgcctc aacagcatca agaagctgtc caccatcgcc ttggcccttg gggttgaaag
     5461 gacccgaagt gagcttctgc ctttccttac agataccatc tatgatgaag atgaggtcct
     5521 cctggccctg gcagaacagc tgggaacctt cactaccctg gtgggaggcc cagagtacgt
     5581 gcactgcctg ctgccaccgc tggagtcgct ggccacagtg gaggagacag tggtgcggga
     5641 caaggcagtg gagtccttac gggccatctc acacgagcac tcgccctctg acctggaggc
     5701 gcactttgtg ccgctagtga agcggctggc gggcggcgac tggttcacct cccgcacctc
     5761 ggcctgcggc ctcttctccg tctgctaccc ccgagtgtcc agtgctgtga aggcggaact
     5821 tcgacagtac ttccggaacc tgtgctcaga tgacaccccc atggtgcggc gggccgcagc
     5881 ctccaagctg ggggagtttg ccaaggtgct ggagctggac aacgtcaaga gtgagatcat
     5941 ccccatgttc tccaacctgg cctctgacga gcaggactcg gtgcggctgc tggcggtgga
     6001 ggcgtgcgtg aacatcgccc agcttctgcc ccaggaggat ctggaggccc tggtgatgcc
     6061 cactctgcgc caggccgctg aagacaagtc ctggcgcgtc cgctacatgg tggctgacaa
     6121 gttcacagag ctccagaaag cagtggggcc tgagatcacc aagacagacc tggtccctgc
     6181 cttccagaac ctgatgaaag actgtgaggc cgaggtgagg gccgcagcct cccacaaggt
     6241 caaagagttc tgtgaaaacc tctcagctga ctgtcgggag aatgtgatca tgtcccagat
     6301 cttgccctgc atcaaggagc tggtgtccga tgccaaccaa catgtcaagt ctgccctggc
     6361 ctcagtcatc atgggtctct ctcccatctt gggcaaagac aacaccatcg agcacctctt
     6421 gcccctcttc ctggctcagc tgaaggatga gtgccctgag gtacggctga acatcatctc
     6481 taacctggac tgtgtgaacg aggtgattgg catccggcag ctgtcccagt ccctgc
//
