LOCUS       sequence-485894-        6687 bp    DNA     circular SYN 12-SEP-2025
DEFINITION  Express CRAF(FL)-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 6687)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6687)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 12, 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            "Unpublished"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, CRAF(FL)-NanoLuc
FEATURES             Location/Qualifiers
     source          1..6687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(166..369)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(195..215)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        370..749
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    940..1223
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     rep_origin      1339..1927
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1828..1847
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    2166..2214
                     /note="synthetic polyadenylation signal"
     CDS             complement(2278..3072)
                     /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(2389..2408)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2999..3018
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3104..3151)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(3166..3523)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3180..3315
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        3332..3523
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     3480..3500
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    3768..3889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3805..3824)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3859..3878
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3926..3944
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4032..4544)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(6522..6541)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6523..6541)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6583..6602)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          join(6585..6687,1..30)
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
ORIGIN
        1 ttaaacctgt cttgtaacct tgatacttac ctgcccagtg cctcacgacc aacttctgca
       61 gcttaagttc gagactgttg tgtcagaagc actgactgcg ttagcaattt aactgtgata
      121 aactaccgca ataaagcttc tagtgatctg acggttcact aaaccagctc tgcttatata
      181 gacctcccac cgtacacgcc taccgcccat ttgcgtcaac ggggcggggt tattacgaca
      241 ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg
      301 gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattggtgt actgccaaaa
      361 ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc
      421 ccgtaaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat
      481 agggggcgga cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa
      541 atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt
      601 cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt
      661 aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta
      721 ctattaataa ctagtcaata atcaatgcca acatggcggt catattggac atgagccaat
      781 ataaatgtac atattatgat atagatacaa cgtatgcaat ggccaatagc caatattgat
      841 ttatgctata taaccaatga ataatatggc taatggccaa tattgaagat ctcgatcctc
      901 tacgccggac gcatcgtggc cggagtactg cggtttatct cgatggctac gagggcagac
      961 agtaagtgga tttaccataa tcccttaatt gtacgcaccg ctaaaacgcg ttcagcgcga
     1021 tcacggcagc agacaggtaa aaatggcaac aaaccacccg aaaaactgcc gcaatcgcgc
     1081 ctgataaatt ttaaccgtat gaatacctat gcaaccagag ggtacaggcc acattacccc
     1141 cacttaatcc actgaagctg ccatttttca tggtttcacc atcccagcga agggccatcc
     1201 agcgtgcgtt cctgtatttc cggactataa aaggatctag gtgaagatcc tttttgataa
     1261 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1321 aaagatcaaa ggatcttctt gaaatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1381 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1441 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     1501 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1561 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1621 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1681 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     1741 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     1801 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     1861 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     1921 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     1981 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     2041 gtgagctgat accgggtaga aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt
     2101 cagctactgg gctatctgga caagggaaaa cgcaagcgca aagaggatcc ttatcgctat
     2161 cgattcacac aaaaaaccaa cacacagatg taatgaaaat aaagatattt tattgcggcc
     2221 atcgtgatgg caggttgggc gtcgcttggt cggtcatttc gaaccccaga gtcccgctca
     2281 gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc
     2341 gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt
     2401 agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc
     2461 agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac
     2521 gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag
     2581 cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg
     2641 tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt
     2701 atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga
     2761 tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt
     2821 gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc
     2881 tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg
     2941 gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc
     3001 ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc
     3061 ttgttcaatc atgatctgcc tcctgggttt agttcctcac cttgtcgtat tatactatgc
     3121 cgatatacta tgccgatgat taattgtcaa cagttaatta agctttttgc aaaagcctag
     3181 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg cggcctcggc
     3241 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     3301 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg
     3361 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga
     3421 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     3481 ccctaactga cacacattcc acagctggtt ctttccgcct cagaaggtac ctaaccaagt
     3541 tcctctttca gaggttattt caggccatgg tgctgcgcag atccgcgtat gcggtgtgaa
     3601 ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc gttaatattt
     3661 tgttaaaatt cgattttacc acatttgtag aggttttact tgcttaaaaa aacctcccac
     3721 atctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     3781 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3841 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgct
     3901 cgaagcggcc gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc
     3961 agcttccttt cgggctttgt tagcagccgg atcagcttgc atgcctgcag gtcgactcta
     4021 gagtcgcggc cttacgccag aatgcgttcg cacagccgcc agccggtcac tccgttgatg
     4081 gttactcgga acagcaggga gccgtcgggg ttgatcaggc gctcgtcgat aattttgttg
     4141 ccgttccaca gggtccctgt tacagtgatc tttttgccgt cgaacacggc gatgccttca
     4201 tacggccgtc cgaaatagtc gatcatgttc ggcgtaaccc cgtcgattac cagtgtgcca
     4261 tagtgcagga tcaccttaaa gtgatgatca tccacagggt acaccacctt aaaaattttt
     4321 tcgatctggc ccatttggtc gccgctcaga ccttcatacg ggatgatgac atggatgtcg
     4381 atcttcagcc cattttcacc gctcaggaca atcctttgga tcggagttac ggacaccccg
     4441 agattctgaa acaaactgga cacacctccc tgttcaagga cttggtccag gttgtagccg
     4501 gctgtctgtc gccagtcccc aacgaaatct tcgagtgtga agacgccgct cgagccgaga
     4561 gaaacgaaga caggcagcct cggggacgtg gtcagcgtgc aagcattgat atcctcagtg
     4621 tgggctgccc gatgcaagga tggctcggaa gcgctccggt tgatcttcgg tagagagtgt
     4681 tggagcagct caatggaaga caggatctgg ggaaaaagag gcctctcttc ctttactttc
     4741 ttcacacagt cagctaccag cctcttcatt gctttggggc agttcttata tagcttacta
     4801 agatctgggg aggcatatcc tcggcccacc atgaagatga tctgatctcg gttgttgatg
     4861 tgagaataag gaagctcccc cgtcatcagt tcatacaata cgatgccata ggagtagaca
     4921 tccgactgga aactgaatgg gttgttatcc tgcattcgga tcacctctgg ggccatccag
     4981 aggacagagc cagtaggttg ttcaacctgc tgagaaccac tccagcgtga ctttactgtt
     5041 gccaaaccaa aatctccaat tttcactgtt aagccttcat ggagaaatat attgttggat
     5101 ttcatgtctc tatggatgat gttctttgca tgcaaatagt ccattccctg agccgtctgc
     5161 cgggcaatgt caattagctg gaacatctga aacttggtct cctggacatg caggtgtttg
     5221 tagaggctgc tgccctcgca ccactgggtc acaattgcca ggttgtcctt tgtcatgtac
     5281 cccatgaaaa gcagaatgtt cacatgccgt gttttgcgca gaacagccac ctcattcctg
     5341 aaggcctgga attgctctgg ggttgggtcg acaaccttta ggatctttac tgcaacatct
     5401 ccgtgccatt tacccttata aacagttcca aaagagcctg acccaatccg agtggacagc
     5461 atcacttcac tggcttctat ttcccaataa tagcttgaat ctctctgtcc acgaggccta
     5521 attttgtttt tctcctgggt cccagatact ggtgcccgct ctctttgtgc tggcacgggg
     5581 gttttcggct gtgaccagcc tgttgggctc agattgttgg ggctactgga cagggctgaa
     5641 ggtgaggctg attcgctgtg acttcgaatt gcatcctcaa tcatcctgct gtccacaggc
     5701 agggtggtgc tgaccatgtg gacattaggt gtggatgtcg acctctgcct ctgggagagg
     5761 gaaccttcag atgagggact ggaggtgtta aaggtgaagg cgtgaggtgt agaatatctg
     5821 tgctgagaac taacaggcat cctggaaaca gactctcgca tacgacgcat agtcaaagaa
     5881 ggtagtgctg ggactccact atcaccaata gtggaatttg gaaacaataa gagttgtctg
     5941 atgttactcc agtccacaca catagtaggt actttggtgc tacagtgctc atgaaatttg
     6001 tagccacaag tctgacatcg aaatccattg agcaggaatt tctgacagat gtcacagaag
     6061 gcaagcttca ggaacgtctt ccgagcaaag ttgtgtgttg tgaggggaac atgatccagg
     6121 aaatctactt gaagttcttc tccaatcaaa gacgcagcat cagtattcca atctaagcgt
     6181 gcttttttac ctttgtgttc gtggagaagt ctgaacactg cacagcactc tggttgcagg
     6241 cccctcacct tgagtgcttt cataaggcag tcatgcaagc tcattccatt tcgcacattg
     6301 accactgttc tttgcttgtt cggcaagaaa acacggatag tgttgcttgt cttagaagga
     6361 tctgtgagtt tgccatcatc tgatgcccgg cgctgatagc caaactgctg aactattgta
     6421 ggagagatgc agctggagcc atcaaacacg gcatctttga atccaaaacc attgctgatc
     6481 gtcttccaag ctccctgtat gtgctccatg gcgatcgcta gccctatagt gagtcgtatt
     6541 aatactctag ccttaagagc tgtaattgaa ctgggagtgg acacctgtgg agagaaaggc
     6601 aaagtggatg tcagtaagac caataggtgc ctatcagaaa cgcaagagtc ttctctgtct
     6661 cgacaagccc agtttctatt ggtctcc
//
