LOCUS       sequence-477411-        8167 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express E6AP-NanoLuc(R) 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 8167)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8167)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: NanoBRET Assay Donor Vector, E6AP
FEATURES             Location/Qualifiers
     source          1..8167
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             34..546
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     polyA_signal    723..804
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(760..779)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        821..1999
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1052..1990
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1947..1967
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2377..3171
                     /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(2431..2450)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3041..3060
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3195..3243
                     /note="synthetic polyadenylation signal"
     rep_origin      3369..3957
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3858..3877
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4129..4788)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4703..4722
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(4820..4867)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(4900..4920)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5075..5378
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5498..5516
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5498..5515
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 tttggcatgc tggtttctct cggctcgagc ggcgtcttca cactcgaaga tttcgttggg
       61 gactggcgac agacagccgg ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc
      121 agtttgtttc agaatctcgg ggtgtccgta actccgatcc aaaggattgt cctgagcggt
      181 gaaaatgggc tgaagatcga catccatgtc atcatcccgt atgaaggtct gagcggcgac
      241 caaatgggcc agatcgaaaa aatttttaag gtggtgtacc ctgtggatga tcatcacttt
      301 aaggtgatcc tgcactatgg cacactggta atcgacgggg ttacgccgaa catgatcgac
      361 tatttcggac ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca
      421 gggaccctgt ggaacggcaa caaaattatc gacgagcgcc tgatcaaccc cgacggctcc
      481 ctgctgttcc gagtaaccat caacggagtg accggctggc ggctgtgcga acgcattctg
      541 gcgtaaggcc gcgactctag aaacacctag gctctcgtac gaagtcagct gataacgtcg
      601 acgtattgcg gccgcggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
      661 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
      721 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
      781 caaataaagc atttttttca ctgccgacgt cacgcgtctt ggctccggtg cccgtcagtg
      841 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
      901 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
      961 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
     1021 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc
     1081 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta
     1141 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc
     1201 ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg
     1261 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat
     1321 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc
     1381 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg
     1441 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg
     1501 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat
     1561 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg
     1621 gccgcttccc ggccctgctg caggcagctc aaaatggagg acgcggcgct cgggagagcg
     1681 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg
     1741 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag
     1801 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg
     1861 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct
     1921 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct
     1981 tccatttcag gtgtcgtgaa agccgaattc tgcagatatc catcacactg tcgagcatgc
     2041 atctagtcca gtgtggtgga attcggcttc gtggggacgc cgtacaggga cgtgcacctc
     2101 tcccgctgca ccgcctccag cgtcgccgcc ggctcgaagg acggggccgg gatgacgatg
     2161 caggcggcgt gggaggtggc gcccaagttg cccatgacca tgccgaagca gtggtagaag
     2221 ggcaccggca gacacacccg gtcctgctcc gtgtagccga ccgtgcggcc cacccagtag
     2281 ccgttgttga ggatgttgtg gtgggagagc gtggcgccct tggggaagcc ggtgtgccgg
     2341 aggtgtactg gatgttgacc gggaatcgat gccaccatga ttgaacaaga tggattgcac
     2401 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     2461 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     2521 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     2581 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     2641 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     2701 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     2761 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     2821 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     2881 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccac
     2941 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3001 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     3061 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     3121 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg aactctagcc
     3181 atcacgatgg ccgcaataaa atatctttat tttcattaca tctgtgtgtt ggttttttgt
     3241 gtgacgattc cggactataa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     3301 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3361 ggatcttctt gaaatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3421 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     3481 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     3541 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     3601 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     3661 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3721 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3781 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3841 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3901 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3961 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4021 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4081 accggttggg cgtcgcttgg tcggtcattt cgaaccccag agtcccgctt acgccccgcc
     4141 ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc
     4201 acagacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata
     4261 atatttgccc atagtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc
     4321 aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc
     4381 tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag
     4441 aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc
     4501 atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat
     4561 tgccatacgg aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg
     4621 ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac
     4681 ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg
     4741 ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccatga tctgcctcct
     4801 gggtttagtt cctcaccttg tcgtattata ctatgccgat atactatgcc gatgattaat
     4861 tgtcaacagt attagaattc gatttgacct cccaccgtac acgcctaccg cccatttgcg
     4921 tcaacggggc ggggttatta cgacattttg gaaagtcccg ttgattttgg tgccaaaaca
     4981 aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca aaccgctatc
     5041 cacgcccatt ggtgtactgc caaaaccgca tcaccatggt aatagcgatg actaatacgt
     5101 agatgtactg ccaagtagga aagtcccgta aggtcatgta ctgggcataa tgccaggcgg
     5161 gccatttacc gtcattgacg tcaatagggg gcggacttgg catatgatac acttgatgta
     5221 ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta
     5281 ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg
     5341 gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata tatgggctat
     5401 gaactaatga ccccgtaatt gattactatt aataactagt ggcgcgccag ggaggcctat
     5461 ataagcagag ctggtttagt gaaccgtcag atcgcccatt taggtgacac tatagaatac
     5521 aagctacttg ttctttttgc agctagcgat cgccatggag aagctgcacc agtgttattg
     5581 gaaatcagga gaacctcagt ctgacgacat tgaagctagc cgaatgaagc gagcagctgc
     5641 aaagcatcta atagaacgct actaccacca gttaactgag ggctgtggaa atgaagcctg
     5701 cacgaatgag ttttgtgctt cctgtccaac ttttcttcgt atggataata atgcagcagc
     5761 tattaaagcc ctcgagcttt ataagattaa tgcaaaactc tgtgatcctc atccctccaa
     5821 gaaaggagca agctcagctt accttgagaa ctcgaaaggt gcccccaaca actcctgctc
     5881 tgagataaaa atgaacaaga aaggcgctag aattgatttt aaagatgtga cttacttaac
     5941 agaagagaag gtatatgaaa ttcttgaatt atgtagagaa agagaggatt attccccttt
     6001 aatccgtgtt attggaagag ttttttctag tgctgaggca ttggtacaga gcttccggaa
     6061 agttaaacaa cacaccaagg aagaactgaa atctcttcaa gcaaaagatg aagacaaaga
     6121 tgaagatgaa aaggaaaaag ctgcatgttc tgctgctgct atggaagaag actcagaagc
     6181 atcttcctca aggataggtg atagctcaca gggagacaac aatttgcaaa aattaggccc
     6241 tgatgatgtg tctgtggata ttgatgccat tagaagggtc tacaccagat tgctctctaa
     6301 tgaaaaaatt gaaactgcct ttctcaatgc acttgtatat ttgtcaccta acgtggaatg
     6361 tgacttgacg tatcacaatg tatactctcg agatcctaat tatctgaatt tgttcattat
     6421 cgtaatggag aatagaaatc tccacagtcc tgaatatctg gaaatggctt tgccattatt
     6481 ttgcaaagcg atgagcaagc taccccttgc agcccaagga aaactgatca gactgtggtc
     6541 taaatacaat gcagaccaga ttcggagaat gatggagaca tttcagcaac ttattactta
     6601 taaagtcata agcaatgaat ttaacagtcg aaatctagtg aatgatgatg atgccattgt
     6661 tgctgcttcg aagtgcttga aaatggttta ctatgcaaat gtagtgggag gggaagtgga
     6721 cacaaatcac aatgaagaag atgatgaaga gcccatccct gagtccagcg agctgacact
     6781 tcaggaactt ttgggagaag aaagaagaaa caagaaaggt cctcgagtgg accccctgga
     6841 aactgaactt ggtgttaaaa ccctggattg tcgaaaacca cttatccctt ttgaagagtt
     6901 tattaatgaa ccactgaatg aggttctaga aatggataaa gattatactt ttttcaaagt
     6961 agaaacagag aacaaattct cttttatgac atgtcccttt atattgaatg ctgtcacaaa
     7021 gaatttggga ttatattatg acaatagaat tcgcatgtac agtgaacgaa gaatcactgt
     7081 tctctacagc ttagttcaag gacagcagtt gaatccatat ttgagactca aagttagacg
     7141 tgaccatatc atagatgatg cacttgtccg gctagagatg atcgctatgg aaaatcctgc
     7201 agacttgaag aagcagttgt atgtggaatt tgaaggagaa caaggagttg atgagggagg
     7261 tgtttccaaa gaattttttc agctggttgt ggaggaaatc ttcaatccag atattggtat
     7321 gttcacatac gatgaatcta caaaattgtt ttggtttaat ccatcttctt ttgaaactga
     7381 gggtcagttt actctgattg gcatagtact gggtctggct atttacaata actgtatact
     7441 ggatgtacat tttcccatgg ttgtctacag gaagctaatg gggaaaaaag gaacttttcg
     7501 tgacttggga gactctcacc cagttctata tcagagttta aaagatttat tggagtatga
     7561 agggaatgtg gaagatgaca tgatgatcac tttccagata tcacagacag atctttttgg
     7621 taacccaatg atgtatgatc taaaggaaaa tggtgataaa attccaatta caaatgaaaa
     7681 caggaaggaa tttgtcaatc tttattctga ctacattctc aataaatcag tagaaaaaca
     7741 gttcaaggct tttcggagag gttttcatat ggtgaccaat gaatctccct taaagtactt
     7801 attcagacca gaagaaattg aattgcttat atgtggaagc cggaatctag atttccaagc
     7861 actagaagaa actacagaat atgacggtgg ctataccagg gactctgttc tgattaggga
     7921 gttctgggaa atcgttcatt catttacaga tgaacagaaa agactcttct tgcagtttac
     7981 aacgggcaca gacagagcac ctgtgggagg actaggaaaa ttaaagatga ttatagccaa
     8041 aaatggccca gacacagaaa ggttacctac atctcatact tgctttaatg tgcttttact
     8101 tccggaatac tcaagcaaag aaaaacttaa agagagattg ttgaaggcca tcacgtatgc
     8161 caaagga
//
