LOCUS       sequence-475272-        6734 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express CRAF-LgBiT 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 6734)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6734)
  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            "Unpublished"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: CMV/CRAF-LgBiT/HygR Vector
FEATURES             Location/Qualifiers
     source          1..6734
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    17..98
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     42..61
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(2788..2806)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2789..2806)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        2926..3229
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3384..3404
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        3437..3484
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3516..4310
                     /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(3570..3589)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4180..4199
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4468..5056)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4548..4567)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5182..5230
                     /note="synthetic polyadenylation signal"
     CDS             complement(5254..6291)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             5267..5275
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     promoter        complement(6322..6679)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6336..6471
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6488..6679
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6636..6656
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 aagacgcgtg acgtcggcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
       61 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
      121 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
      181 aatgtggtat ggctgattat gatccgcggc cgcaatacgt cgacgttatc agctgacttc
      241 gtacgagagc ctaggtgttt ctagagtcgc ggccttagct gttgatggtt actcggaaca
      301 gcatggagcc gtcgggggtg atcaggcgct cgtcgataat tttgttgccg ttccacaggg
      361 tccctgttac agtgatcttt ttgccgtcga acacggcgat gccttcatac ggccgtccga
      421 aatagttcag catgttcggc gtaaccccgt cgattaccag tgtgccatag ggcaggatca
      481 ccttaaagtg atgatcatcc acagggtaca ccaccttaaa cacctcttcg atctgggcca
      541 tttggtcggc gctcagacct tcatacggga tgatgacatg gatgtcgatc ttcagggcat
      601 tttcaccgct ccggacaatc ctttggatcg gagttacgga cacggcgaga ttctgcagca
      661 aactggacac acctccctgt tcaaggactt ggtccaggtt gtaggcggct gtctgttccc
      721 agtccccaac gaaatcttcg agtgtgaaga caccgctcga gcctccacct ccgctcccgc
      781 caccacctga actgccttga gaaacgaaga caggcagcct cggggacgtg gtcagcgtgc
      841 aagcattgat atcctcagtg tgggctgccc gatgcaagga tggctcggaa gcgctccggt
      901 tgatcttcgg tagagagtgt tggagcagct caatggaaga caggatctgg ggaaaaagag
      961 gcctctcttc ctttactttc ttcacacagt cagctaccag cctcttcatt gctttggggc
     1021 agttcttata tagcttacta agatctgggg aggcatatcc tcggcccacc atgaagatga
     1081 tctgatctcg gttgttgatg tgagaataag gaagctcccc cgtcatcagt tcatacaata
     1141 cgatgccata ggagtagaca tccgactgga aactgaatgg gttgttatcc tgcattcgga
     1201 tcacctctgg ggccatccag aggacagagc cagtaggttg ttcaacctgc tgagaaccac
     1261 tccagcgtga ctttactgtt gccaaaccaa aatctccaat tttcactgtt aagccttcat
     1321 ggagaaatat attgttggat ttcatgtctc tatggatgat gttctttgca tgcaaatagt
     1381 ccattccctg agccgtctgc cgggcaatgt caattagctg gaacatctga aacttggtct
     1441 cctggacatg caggtgtttg tagaggctgc tgccctcgca ccactgggtc acaattgcca
     1501 ggttgtcctt tgtcatgtac cccatgaaaa gcagaatgtt cacatgccgt gttttgcgca
     1561 gaacagccac ctcattcctg aaggcctgga attgctctgg ggttgggtcg acaaccttta
     1621 ggatctttac tgcaacatct ccgtgccatt tacccttata aacagttcca aaagagcctg
     1681 acccaatccg agtggacagc atcacttcac tggcttctat ttcccaataa tagcttgaat
     1741 ctctctgtcc acgaggccta attttgtttt tctcctgggt cccagatact ggtgcccgct
     1801 ctctttgtgc tggcacgggg gttttcggct gtgaccagcc tgttgggctc agattgttgg
     1861 ggctactgga cagggctgaa ggtgaggctg attcgctgtg acttcgaatt gcatcctcaa
     1921 tcatcctgct gtccacaggc agggtggtgc tgaccatgtg gacattaggt gtggatgtcg
     1981 acctctgcct ctgggagagg gaaccttcag atgagggact ggaggtgtta aaggtgaagg
     2041 cgtgaggtgt agaatatctg tgctgagaac taacaggcat cctggaaaca gactctcgca
     2101 tacgacgcat agtcaaagaa ggtagtgctg ggactccact atcaccaata gtggaatttg
     2161 gaaacaataa gagttgtctg atgttactcc agtccacaca catagtaggt actttggtgc
     2221 tacagtgctc atgaaatttg tagccacaag tctgacatcg aaatccattg agcaggaatt
     2281 tctgacagat gtcacagaag gcaagcttca ggaacgtctt ccgagcaaag ttgtgtgttg
     2341 tgaggggaac atgatccagg aaatctactt gaagttcttc tccaatcaaa gacgcagcat
     2401 cagtattcca atctaagcgt gcttttttac ctttgtgttc gtggagaagt ctgaacactg
     2461 cacagcactc tggttgcagg cccctcacct tgagtgcttt cataaggcag tcatgcaagc
     2521 tcattccatt tcgcacattg accactgttc tttgcttgtt cggcaagaaa acacggatag
     2581 tgttgcttgt cttagaagga tctgtgagtt tgccatcatc tgatgcccgg cgctgatagc
     2641 caaactgctg aactattgta ggagagatgc agctggagcc atcaaacacg gcatctttga
     2701 atccaaaacc attgctgatc gtcttccaag ctccctgtat gtgctccatg gcgatcgcta
     2761 gctgcaaaaa gaacaagtag cttgtattct atagtgtcac ctaaatgggc gatctgacgg
     2821 ttcactaaac cagctctgct tatataggcc tccctggcgc gccactagtt attaatagta
     2881 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     2941 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     3001 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     3061 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat
     3121 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga
     3181 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     3241 ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     3301 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     3361 tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt gggaggtcaa
     3421 atcgaattct aatactgttg acaattaatc atcggcatag tatatcggca tagtataata
     3481 cgacaaggtg aggaactaaa cccaggaggc agatcatgat tgaacaagat ggattgcacg
     3541 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3601 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     3661 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     3721 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     3781 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     3841 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     3901 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     3961 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     4021 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     4081 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4141 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4201 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4261 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga ttcgaaatga
     4321 ccgaccaagc gacgcccaac cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4381 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4441 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4501 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4561 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4621 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4681 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4741 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4801 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4861 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     4921 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4981 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5041 aaaaaaagga tttcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5101 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5161 ccttttatag tccggaatcg tcacacaaaa aaccaacaca cagatgtaat gaaaataaag
     5221 atattttatt gcggccatcg tgatggctag agtctaaact cgacctacct ccttagcgcg
     5281 cggtcgtgta ctgggccggc ggttgccgct gtcggccagc acctcgacgc agccgtcggt
     5341 ccatacggct gcgctccggc gagcgatttg tgtgcgaccg acggtgccgg ccccgctgcg
     5401 gacgatggca tcgcagcggc cttgagccca ggcagcatcg tcgaagttgc cgtccacgag
     5461 gctctggtag agctgatcca ggccgatgcg cagcatgtag gctcgcagac gagggctgcc
     5521 ggccagctcg ggatgccggc gctcgaagta gcgagtctgc tgctccatgc aagccagcca
     5581 gggccgccag aagaagatgt tggccacctc gtactgactg tccccgaaca tagcttcgga
     5641 ccagtcgatt acggcggtga tgcggccgtt gtcggtcagg acgttgttgc tgccgaagtc
     5701 ggcatggacg aggtggcgca cctcgggaca gtcttcggcc cacagcatca gttcgtccag
     5761 ggcttgagct acgctggcgg acacggtgtc gtccatcacg gtctgccagt ggtagacatg
     5821 gggatcagca atggcgcaaa tgaaatcccg ccaggttgtg tactggccga tgccttgggg
     5881 cccgaatggg ccgaagccgc tggtttgact gaggtcggcg gcggcaatag catccatagc
     5941 ttcggcgaca ggctgtaaca cagcaggcag ctctgtttcg gggaggtctt ggagagtaac
     6001 gccttgggcg cgtctactga tgcagtatgt caggctctcg ctgaactcgc cgatgtccaa
     6061 cacttcgggg atgggtagtg cagcgctggc gaagtggcgg tacacgtagc ggtctttgta
     6121 gaagccatca gcgcagctat tcacccgcag tacatagccg cgtccgccga catcgaagct
     6181 gaaggctcgg ctctcttcgc cctccgacaa ctgcatcagg tcgctcacac tgtcgaactt
     6241 ctcgatgaga aatttttcaa cgctggtagc ggtgagttcg ggcttcttca tggtggcgct
     6301 agtgtcagaa gaatcgagct ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta
     6361 cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa aaaaaattag
     6421 tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt
     6481 taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc
     6541 tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca tgctttgcat
     6601 acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca cattccacag
     6661 ctggttcttt ccgcctcaga aggtacctaa ccaagttcct ctttcagagg ttatttcagg
     6721 ccatggtgct gcgc
//
