LOCUS       sequence-486147-        6561 bp    DNA     circular SYN 25-SEP-2025
DEFINITION  Express ARAF-NanoLuc(R) in Mammalian Cells under a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6561)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6561)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 25, 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            "Unpublished"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, ARAF-NanoLuc
FEATURES             Location/Qualifiers
     source          1..6561
                     /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"
     CDS             2904..3416
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(3504..3522)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3559..3680
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3570..3589)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3624..3643
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3925..4282
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3925..4116
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3948..3968)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4133..4268
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4297..4344
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4376..5170
                     /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(4430..4449)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5040..5059
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5234..5282
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(5521..6109)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5601..5620)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6225..6508
                     /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 cgccatggag ccaccacggg
     1081 gcccccctgc caatggggcc gagccatccc gggcagtggg caccgtcaaa gtatacctgc
     1141 ccaacaagca acgcacggtg gtgactgtcc gggatggcat gagtgtctac gactctctag
     1201 acaaggccct gaaggtgcgg ggtctaaatc aggactgctg tgtggtctac cgactcatca
     1261 agggacgaaa gacggtcact gcctgggaca cagccattgc tcccctggat ggcgaggagc
     1321 tcattgtcga ggtccttgaa gatgtcccgc tgaccatgca caattttgta cggaagacct
     1381 tcttcagcct ggcgttctgt gacttctgcc ttaagtttct gttccatggc ttccgttgcc
     1441 aaacctgtgg ctacaagttc caccagcatt gttcctccaa ggtccccaca gtctgtgttg
     1501 acatgagtac caaccgccaa cagttctacc acagtgtcca ggatttgtcc ggaggctcca
     1561 gacagcatga ggctccctcg aaccgccccc tgaatgagtt gctaaccccc cagggtccca
     1621 gcccccgcac ccagcactgt gacccggagc acttcccctt ccctgcccca gccaatgccc
     1681 ccctacagcg catccgctcc acgtccactc ccaacgtcca tatggtcagc accacggccc
     1741 ccatggactc caacctcatc cagctcactg gccagagttt cagcactgat gctgccggta
     1801 gtagaggagg tagtgatgga accccccggg ggagccccag cccagccagc gtgtcctcgg
     1861 ggaggaagtc cccacattcc aagtcaccag cagagcagcg cgagcggaag tccttggccg
     1921 atgacaagaa gaaagtgaag aacctggggt accgggactc aggctattac tgggaggtac
     1981 cacccagtga ggtgcagctg ctgaagagga tcgggacggg ctcgtttggc accgtgtttc
     2041 gagggcggtg gcatggcgat gtggccgtga aggtgctcaa ggtgtcccag cccacagctg
     2101 agcaggccca ggctttcaag aatgagatgc aggtgctcag gaagacgcga catgtcaaca
     2161 tcttgctgtt tatgggcttc atgacccggc cgggatttgc catcatcaca cagtggtgtg
     2221 agggctccag cctctaccat cacctgcatg tggccgacac acgcttcgac atggtccagc
     2281 tcatcgacgt ggcccggcag actgcccagg gcatggacta cctccatgcc aagaacatca
     2341 tccaccgaga tctcaagtct aacaacatct tcctacatga ggggctcacg gtgaagatcg
     2401 gtgactttgg cttggccaca gtgaagactc gatggagcgg ggcccagccc ttggagcagc
     2461 cctcaggatc tgtgctgtgg atggcagctg aggtgatccg tatgcaggac ccgaacccct
     2521 acagcttcca gtcagacgtc tatgcctacg gggttgtgct ctacgagctt atgactggct
     2581 cactgcctta cagccacatt ggctgccgtg accagattat ctttatggtg ggccgtggct
     2641 atctgtcccc ggacctcagc aaaatctcca gcaactgccc caaggccatg cggcgcctgc
     2701 tgtctgactg cctcaagttc cagcgggagg agcggcccct cttcccccag atcctggcca
     2761 caattgagct gctgcaacgg tcactcccca agattgagcg gagtgcctcg gaaccctcct
     2821 tgcaccgcac ccaggccgat gagttgcctg cctgcctact cagcgcagcc cgccttgtgc
     2881 ctgtttctct cggctcgagc ggcgtcttca cactcgaaga tttcgttggg gactggcgac
     2941 agacagccgg ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc
     3001 agaatctcgg ggtgtccgta actccgatcc aaaggattgt cctgagcggt gaaaatgggc
     3061 tgaagatcga catccatgtc atcatcccgt atgaaggtct gagcggcgac caaatgggcc
     3121 agatcgaaaa aatttttaag gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc
     3181 tgcactatgg cacactggta atcgacgggg ttacgccgaa catgatcgac tatttcggac
     3241 ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt
     3301 ggaacggcaa caaaattatc gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc
     3361 gagtaaccat caacggagtg accggctggc ggctgtgcga acgcattctg gcgtaaggcc
     3421 gcgactctag agtcgacctg caggcatgca agctgatccg gctgctaaca aagcccgaaa
     3481 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcggc
     3541 cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     3601 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     3661 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     3721 gggagatgtg ggaggttttt ttaagcaagt aaaacctcta caaatgtggt aaaatcgaat
     3781 tttaacaaaa tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt
     3841 gcggtatttc acaccgcata cgcggatctg cgcagcacca tggcctgaaa taacctctga
     3901 aagaggaact tggttaggta ccttctgagg cggaaagaac cagctgtgga atgtgtgtca
     3961 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4021 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     4081 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     4141 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4201 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     4261 tggaggccta ggcttttgca aaaagcttaa ttaactgttg acaattaatc atcggcatag
     4321 tatatcggca tagtataata cgacaaggtg aggaactaaa cccaggaggc agatcatgat
     4381 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4441 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4501 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga
     4561 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4621 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4681 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4741 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4801 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4861 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga
     4921 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4981 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     5041 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     5101 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     5161 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     5221 tcacgatggc cgcaataaaa tatctttatt ttcattacat ctgtgtgttg gttttttgtg
     5281 tgaatcgata gcgataagga tcctctttgc gcttgcgttt tcccttgtcc agatagccca
     5341 gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct acccggtatc
     5401 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5461 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5521 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5581 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5641 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5701 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5761 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5821 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5881 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5941 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     6001 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6061 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatttcaag aagatccttt
     6121 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6181 catgagatta tcaaaaagga tcttcaccta gatcctttta tagtccggaa atacaggaac
     6241 gcacgctgga tggcccttcg ctgggatggt gaaaccatga aaaatggcag cttcagtgga
     6301 ttaagtgggg gtaatgtggc ctgtaccctc tggttgcata ggtattcata cggttaaaat
     6361 ttatcaggcg cgattgcggc agtttttcgg gtggtttgtt gccattttta cctgtctgct
     6421 gccgtgatcg cgctgaacgc gttttagcgg tgcgtacaat taagggatta tggtaaatcc
     6481 acttactgtc tgccctcgta gccatcgaga taaaccgcag tactccggcc acgatgcgtc
     6541 cggcgtagag gatcgagatc t
//
