LOCUS       sequence-471924-        4722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express CRAF(RBD-CRD)-SmBiT 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 4722)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4722)
  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            "Unpublished"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: CRAF(RBD-CRD)-SmBiT BiBiT-Ready Vector
FEATURES             Location/Qualifiers
     source          1..4722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        338..641
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        642..845
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     796..816
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          981..1113
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1096..1115
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1157..1176
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1157..1175
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1785..1906
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1796..1815)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(2021..2054)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2064..2111
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2143..2937
                     /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(2197..2216)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2807..2826
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(3095..3683)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3175..3194)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3809..3857
                     /note="synthetic polyadenylation signal"
     CDS             complement(3881..4279)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      4276..4285
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4310..4667)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4324..4459
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        4476..4667
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     4624..4644
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 aagacgcgtt aattaagacg tcggcagtga aaaaaatgct ttatttgtga aatttgtgat
       61 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
      121 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
      181 ctctacaaat gtggtatggc tgattatgat ccgcggccgc aatacgtcga cgttatcagc
      241 tgacttcgta cgagagccta ggattatggc gcgccactag ttattaatag taatcaatta
      301 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      361 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      421 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      481 ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca
      541 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta
      601 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      661 acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      721 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata
      781 accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      841 gagctggttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt tatcacagtt
      901 aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc tgcagaagtt
      961 ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat
     1021 agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc
     1081 ttactgacat ccactttgcc tttctctcca caggtgtcca ctcccagttc aattacagct
     1141 cttaaggcta gagtattaat acgactcact atagggctag cgatcgccat gccttctaag
     1201 acaagcaaca ctatccgtgt tttcttgccg aacaagcaaa gaacagtggt caatgtgcga
     1261 aatggaatga gcttgcatga ctgccttatg aaagcactca aggtgagggg cctgcaacca
     1321 gagtgctgtg cagtgttcag acttctccac gaacacaaag gtaaaaaagc acgcttagat
     1381 tggaatactg atgctgcgtc tttgattgga gaagaacttc aagtagattt cctggatcat
     1441 gttcccctca caacacacaa ctttgctcgg aagacgttcc tgaagcttgc cttctgtgac
     1501 atctgtcaga aattcctgct caatggattt cgatgtcaga cttgtggcta caaatttcat
     1561 gagcactgta gcaccaaagt acctactatg tgtgtggact ggagtaacgt ttctcaaggc
     1621 agttcaggtg gtggcgggag cggaggtgga ggctcgagcg gtgtgaccgg ctaccggctg
     1681 ttcgaggaga ttctgtaagg ccgcgactct agagtcgacc tgcaggcatg caagctgatt
     1741 tggctgctgc caccgctgag caaggccgct tcgagcagac atgataagat acattgatga
     1801 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     1861 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     1921 cattcatttt atgtttcagg ttcaggggga gatgtgggag gtttttttaa gcaagtaaaa
     1981 cctctacaaa tgtggtaaaa tcttaattaa gaattctaat ataacttcgt atagcataca
     2041 ttatacgaag ttatggatcc actgttgaca attaatcatc ggcatagtat atcggcatag
     2101 tataatacga caaggtgagg aactaaaccc aggaggcaga tcatgattga acaagatgga
     2161 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     2221 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     2281 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     2341 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     2401 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     2461 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     2521 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     2581 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     2641 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     2701 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     2761 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     2821 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     2881 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgattc
     2941 gaaatgaccg accaagcgac gcccaaccgg tatcagctca ctcaaaggcg gtaatacggt
     3001 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3061 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3121 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3181 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3241 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3301 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3361 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3421 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     3481 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     3541 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3601 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3661 cgcgcagaaa aaaaggattt caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3721 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3781 cctagatcct tttatagtcc ggaatcgtca cacaaaaaac caacacacag atgtaatgaa
     3841 aataaagata ttttattgcg gccatcgtga tggctagagt ttagccctcc cacacataac
     3901 cagagggcag caattcacga atcccaactg ccgtcggctg tccatcactg tccttcacta
     3961 tggctttgat cccaggatgc agatcgagaa gcacctgtcg gcaccgtccg caggggctca
     4021 agatgcccct gttctcattt ccgatcgcga cgatacaagt caggttgcca gctgccgcag
     4081 cagcagcagt gcccagcacc acgagttctg cacaaggtcc cccagtaaaa tgatatacat
     4141 tgacaccagt gaagatgcgg ccgtcgctag agagagctgc gctggcgacg ctgtagtctt
     4201 cagagatggg gatgctgttg attgtagccg ttgctctttc aatgagggtg gattcttctt
     4261 gagacaaagg cttggccatg gtggcgctag tgtcagaaga atcgagcttt ttgcaaaagc
     4321 ctaggcctcc aaaaaagcct cctcactact tctggaatag ctcagaggcc gaggcggcct
     4381 cggcctctgc ataaataaaa aaaattagtc agccatgggg cggagaatgg gcggaactgg
     4441 gcggagttag gggcgggatg ggcggagtta ggggcgggac tatggttgct gactaattga
     4501 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca cacctggttg
     4561 ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc
     4621 cacaccctaa ctgacacaca ttccacagct ggttctttcc gcctcagaag gtacctaacc
     4681 aagttcctct ttcagaggtt atttcaggcc atggtgctgc gc
//
