LOCUS       sequence-486133-        6687 bp    DNA     circular SYN 24-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  JOURNAL   Unpublished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 24, 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            "Unpublished"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"
     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             3030..3542
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(3630..3648)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3685..3806
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3696..3715)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3750..3769
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4051..4408
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4051..4242
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4074..4094)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4259..4394
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4423..4470
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4502..5296
                     /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(4556..4575)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5166..5185
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5360..5408
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(5647..6235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5727..5746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6351..6634
                     /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 cacatacagg
     1081 gagcttggaa gacgatcagc aatggttttg gattcaaaga tgccgtgttt gatggctcca
     1141 gctgcatctc tcctacaata gttcagcagt ttggctatca gcgccgggca tcagatgatg
     1201 gcaaactcac agatccttct aagacaagca acactatccg tgttttcttg ccgaacaagc
     1261 aaagaacagt ggtcaatgtg cgaaatggaa tgagcttgca tgactgcctt atgaaagcac
     1321 tcaaggtgag gggcctgcaa ccagagtgct gtgcagtgtt cagacttctc cacgaacaca
     1381 aaggtaaaaa agcacgctta gattggaata ctgatgctgc gtctttgatt ggagaagaac
     1441 ttcaagtaga tttcctggat catgttcccc tcacaacaca caactttgct cggaagacgt
     1501 tcctgaagct tgccttctgt gacatctgtc agaaattcct gctcaatgga tttcgatgtc
     1561 agacttgtgg ctacaaattt catgagcact gtagcaccaa agtacctact atgtgtgtgg
     1621 actggagtaa catcagacaa ctcttattgt ttccaaattc cactattggt gatagtggag
     1681 tcccagcact accttctttg actatgcgtc gtatgcgaga gtctgtttcc aggatgcctg
     1741 ttagttctca gcacagatat tctacacctc acgccttcac ctttaacacc tccagtccct
     1801 catctgaagg ttccctctcc cagaggcaga ggtcgacatc cacacctaat gtccacatgg
     1861 tcagcaccac cctgcctgtg gacagcagga tgattgagga tgcaattcga agtcacagcg
     1921 aatcagcctc accttcagcc ctgtccagta gccccaacaa tctgagccca acaggctggt
     1981 cacagccgaa aacccccgtg ccagcacaaa gagagcgggc accagtatct gggacccagg
     2041 agaaaaacaa aattaggcct cgtggacaga gagattcaag ctattattgg gaaatagaag
     2101 ccagtgaagt gatgctgtcc actcggattg ggtcaggctc ttttggaact gtttataagg
     2161 gtaaatggca cggagatgtt gcagtaaaga tcctaaaggt tgtcgaccca accccagagc
     2221 aattccaggc cttcaggaat gaggtggctg ttctgcgcaa aacacggcat gtgaacattc
     2281 tgcttttcat ggggtacatg acaaaggaca acctggcaat tgtgacccag tggtgcgagg
     2341 gcagcagcct ctacaaacac ctgcatgtcc aggagaccaa gtttcagatg ttccagctaa
     2401 ttgacattgc ccggcagacg gctcagggaa tggactattt gcatgcaaag aacatcatcc
     2461 atagagacat gaaatccaac aatatatttc tccatgaagg cttaacagtg aaaattggag
     2521 attttggttt ggcaacagta aagtcacgct ggagtggttc tcagcaggtt gaacaaccta
     2581 ctggctctgt cctctggatg gccccagagg tgatccgaat gcaggataac aacccattca
     2641 gtttccagtc ggatgtctac tcctatggca tcgtattgta tgaactgatg acgggggagc
     2701 ttccttattc tcacatcaac aaccgagatc agatcatctt catggtgggc cgaggatatg
     2761 cctccccaga tcttagtaag ctatataaga actgccccaa agcaatgaag aggctggtag
     2821 ctgactgtgt gaagaaagta aaggaagaga ggcctctttt tccccagatc ctgtcttcca
     2881 ttgagctgct ccaacactct ctaccgaaga tcaaccggag cgcttccgag ccatccttgc
     2941 atcgggcagc ccacactgag gatatcaatg cttgcacgct gaccacgtcc ccgaggctgc
     3001 ctgtcttcgt ttctctcggc tcgagcggcg tcttcacact cgaagatttc gttggggact
     3061 ggcgacagac agccggctac aacctggacc aagtccttga acagggaggt gtgtccagtt
     3121 tgtttcagaa tctcggggtg tccgtaactc cgatccaaag gattgtcctg agcggtgaaa
     3181 atgggctgaa gatcgacatc catgtcatca tcccgtatga aggtctgagc ggcgaccaaa
     3241 tgggccagat cgaaaaaatt tttaaggtgg tgtaccctgt ggatgatcat cactttaagg
     3301 tgatcctgca ctatggcaca ctggtaatcg acggggttac gccgaacatg atcgactatt
     3361 tcggacggcc gtatgaaggc atcgccgtgt tcgacggcaa aaagatcact gtaacaggga
     3421 ccctgtggaa cggcaacaaa attatcgacg agcgcctgat caaccccgac ggctccctgc
     3481 tgttccgagt aaccatcaac ggagtgaccg gctggcggct gtgcgaacgc attctggcgt
     3541 aaggccgcga ctctagagtc gacctgcagg catgcaagct gatccggctg ctaacaaagc
     3601 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     3661 ggcggccgct tcgagcagac atgataagat acattgatga gtttggacaa accacaacta
     3721 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     3781 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     3841 ttcaggggga gatgtgggag gtttttttaa gcaagtaaaa cctctacaaa tgtggtaaaa
     3901 tcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt ctccttacgc
     3961 atctgtgcgg tatttcacac cgcatacgcg gatctgcgca gcaccatggc ctgaaataac
     4021 ctctgaaaga ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt
     4081 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     4141 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     4201 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     4261 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     4321 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     4381 cttttttgga ggcctaggct tttgcaaaaa gcttaattaa ctgttgacaa ttaatcatcg
     4441 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccca ggaggcagat
     4501 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4561 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4621 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4681 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4741 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4801 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4861 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4921 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4981 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     5041 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     5101 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     5161 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     5221 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     5281 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     5341 ctgccatcac gatggccgca ataaaatatc tttattttca ttacatctgt gtgttggttt
     5401 tttgtgtgaa tcgatagcga taaggatcct ctttgcgctt gcgttttccc ttgtccagat
     5461 agcccagtag ctgacattca tccggggtca gcaccgtttc tgcggactgg ctttctaccc
     5521 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5581 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5641 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5701 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5761 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5821 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5881 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5941 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6001 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6061 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     6121 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6181 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ttcaagaaga
     6241 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6301 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttatagt ccggaaatac
     6361 aggaacgcac gctggatggc ccttcgctgg gatggtgaaa ccatgaaaaa tggcagcttc
     6421 agtggattaa gtgggggtaa tgtggcctgt accctctggt tgcataggta ttcatacggt
     6481 taaaatttat caggcgcgat tgcggcagtt tttcgggtgg tttgttgcca tttttacctg
     6541 tctgctgccg tgatcgcgct gaacgcgttt tagcggtgcg tacaattaag ggattatggt
     6601 aaatccactt actgtctgcc ctcgtagcca tcgagataaa ccgcagtact ccggccacga
     6661 tgcgtccggc gtagaggatc gagatct
//
