LOCUS       sequence-479837-        7088 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express BRAF-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 7088)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7088)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: CMV/BRAF-LgBiT/HygR Vector
FEATURES             Location/Qualifiers
     source          1..7088
                     /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"
     regulatory      3096..3105
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3142..3160)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3143..3160)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        3280..3583
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3738..3758
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        3791..3838
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3870..4664
                     /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(3924..3943)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4534..4553
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4822..5410)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4902..4921)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5536..5584
                     /note="synthetic polyadenylation signal"
     CDS             complement(5608..6645)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             5621..5629
                     /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(6676..7033)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6690..6825
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6842..7033
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6990..7010
                     /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 gaaacgtgga caggaaacgc accatatccc cctgcctgga
      841 tgggtgtttt tggagaagca caagcatata gactaaaatc ctctgtttgg aaaccagccc
      901 gattcaagga gggttctgat gcactgcggt gaatttttgg caatgagcgg gccagcagct
      961 caatagaggc gagaatttgg ggaaagagtg gtctctcatc tcttttcttt ttgaggcact
     1021 ctgccattaa tctcttcatg gcttttggac agttactccg taccttactg agatctggag
     1081 acaggtatcc tcgtcccacc ataaaaatta tctggtccct gttgttgatg tttgaataag
     1141 gtaactgtcc agtcatcaat tcatacagaa caattccaaa tgcatataca tctgactgaa
     1201 agctgtatgg atttttatct tgcattctga tgacttctgg tgccatccac aaaatggatc
     1261 cagacaactg ttcaaactga tgggacccac tccatcgaga tttcactgta gctagaccaa
     1321 aatcacctat ttttactgtg aggtcttcat gaagaaatat attattactc ttgaggtctc
     1381 tgtggatgat tgacttggcg tgtaagtaat ccatgccctg tgcagtctgt cgtgcaatat
     1441 ctataagttt gatcatctca aatttggtct caatgatatg gagatggtga tacaagctgg
     1501 agccctcaca ccactgggta acaatagcca gttgtggctt tgtggaatag cccatgaaga
     1561 gtaggatatt cacatgtcgt gttttcctga gtactcctac ttcatttttg aaggcttgta
     1621 actgctgagg tgtaggtgct gtcacattca acattttcac tgccacatca ccatgccact
     1681 ttcccttgta gactgttcca aatgatccag atccaattct ttgtcccact gtaatctgcc
     1741 catcaggaat ctcccaatca tcactcgagt cccgtctacc aagtgttttc attcgattcc
     1801 tgtcttctga ggatgaagat gacttccttt ctcgctgagg tcctggagat ttctgtaagg
     1861 ctttcacgtt agttagtgag ccaggtaatg aggcaggggg ggtagcagac aaacctgtgg
     1921 ttgatcctcc atcaccacga aatccttggt ctctaatcaa gtcatcaata ttgacaggtt
     1981 ctattgtgtt tatatgcaca ttgggagctg atgaggatcg gtctcgttgc ccaaattgat
     2041 ttcgatgatc ttcatctgct ggtcggaagg gctgtggaat tggaatggat tttgaaggag
     2101 acggactggt gagaatttgg ggcccaatag agtccgaggc gggtgcggaa ggggatgatc
     2161 cagatgttag ggcagtctct gctaaggacg cctcttcctg tggtattggg tggtgttcaa
     2221 agaacttgga gacaaacagc aaatcaagtt ggtcataatt aacacacatc agtggaactt
     2281 ctgtactaca acgctggtga aatttataac cacatgtttg acagcggaaa ccctggaaaa
     2341 gcagctttcg acaaaagtca caaaatgcta aggtgaaaaa cgtttttcgt acaaagttgt
     2401 gtgttgtaag tggaacattc tccaacactt ccacatgcaa ttcttctcca gtaagccagg
     2461 aaatatcagt gtcccaacca attggtttct tctctccatc ctgaattctg taaacagcac
     2521 agcactctgg gattagacct ctcatcatca gtgctttctt tagactgtct cggactgtaa
     2581 ctccacacct tgcaggtacc actgtcctct gtttgttggg caggaagact ctaacgatag
     2641 gtttttgtgg tgacttgggg ttgctccgtg ccacatctgt gggattttga aaaactgaaa
     2701 gagatgaagg tagcactgaa aggctagaag aggaagaaga tgtaacggta tccattgatg
     2761 cagagctaga aacagaaaaa tcagttccgt tccccagaga ttccaataac tgttgttctc
     2821 tttgttggag tgcatctagc ttgctggtgt attcttcata ggcctccaga tatattgatg
     2881 gtggattatg ctccccacca aatttgtcca atagggcctc tatatgttcc tgtgtcaact
     2941 taatcatttg tttgatattc cacacctcct ccggaatggc agggtccgca gccgaagagg
     3001 ccgcggcgcc ggcgccggcg ccggcctcgg gctccatgtc cccgttgaac agagcctggc
     3061 ccggctccgc gccgccacca ccgccaccgc tcagcgccgc catggcgatc gctagctgca
     3121 aaaagaacaa gtagcttgta ttctatagtg tcacctaaat gggcgatctg acggttcact
     3181 aaaccagctc tgcttatata ggcctccctg gcgcgccact agttattaat agtaatcaat
     3241 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     3301 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     3361 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     3421 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt
     3481 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc
     3541 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     3601 gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     3661 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     3721 taaccccgcc ccgttgacgc aaatgggcgg taggcgtgta cggtgggagg tcaaatcgaa
     3781 ttctaatact gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa
     3841 ggtgaggaac taaacccagg aggcagatca tgattgaaca agatggattg cacgcaggtt
     3901 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3961 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     4021 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     4081 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     4141 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     4201 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     4261 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     4321 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     4381 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     4441 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     4501 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     4561 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4621 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgattcgaa atgaccgacc
     4681 aagcgacgcc caaccggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4741 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4801 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4861 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4921 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4981 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5041 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5101 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5161 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5221 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     5281 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5341 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5401 aggatttcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5461 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5521 atagtccgga atcgtcacac aaaaaaccaa cacacagatg taatgaaaat aaagatattt
     5581 tattgcggcc atcgtgatgg ctagagtcta aactcgacct acctccttag cgcgcggtcg
     5641 tgtactgggc cggcggttgc cgctgtcggc cagcacctcg acgcagccgt cggtccatac
     5701 ggctgcgctc cggcgagcga tttgtgtgcg accgacggtg ccggccccgc tgcggacgat
     5761 ggcatcgcag cggccttgag cccaggcagc atcgtcgaag ttgccgtcca cgaggctctg
     5821 gtagagctga tccaggccga tgcgcagcat gtaggctcgc agacgagggc tgccggccag
     5881 ctcgggatgc cggcgctcga agtagcgagt ctgctgctcc atgcaagcca gccagggccg
     5941 ccagaagaag atgttggcca cctcgtactg actgtccccg aacatagctt cggaccagtc
     6001 gattacggcg gtgatgcggc cgttgtcggt caggacgttg ttgctgccga agtcggcatg
     6061 gacgaggtgg cgcacctcgg gacagtcttc ggcccacagc atcagttcgt ccagggcttg
     6121 agctacgctg gcggacacgg tgtcgtccat cacggtctgc cagtggtaga catggggatc
     6181 agcaatggcg caaatgaaat cccgccaggt tgtgtactgg ccgatgcctt ggggcccgaa
     6241 tgggccgaag ccgctggttt gactgaggtc ggcggcggca atagcatcca tagcttcggc
     6301 gacaggctgt aacacagcag gcagctctgt ttcggggagg tcttggagag taacgccttg
     6361 ggcgcgtcta ctgatgcagt atgtcaggct ctcgctgaac tcgccgatgt ccaacacttc
     6421 ggggatgggt agtgcagcgc tggcgaagtg gcggtacacg tagcggtctt tgtagaagcc
     6481 atcagcgcag ctattcaccc gcagtacata gccgcgtccg ccgacatcga agctgaaggc
     6541 tcggctctct tcgccctccg acaactgcat caggtcgctc acactgtcga acttctcgat
     6601 gagaaatttt tcaacgctgg tagcggtgag ttcgggcttc ttcatggtgg cgctagtgtc
     6661 agaagaatcg agctttttgc aaaagcctag gcctccaaaa aagcctcctc actacttctg
     6721 gaatagctca gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc
     6781 atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg
     6841 cgggactatg gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga
     6901 gcctggggac tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct
     6961 gcctgctggg gagcctgggg actttccaca ccctaactga cacacattcc acagctggtt
     7021 ctttccgcct cagaaggtac ctaaccaagt tcctctttca gaggttattt caggccatgg
     7081 tgctgcgc
//
