LOCUS       sequence-474104-        5857 bp    DNA     circular SYN 30-MAY-2025
DEFINITION  Express LgBiT-KRAS (WT)_SmBiT-CRAF-RBD 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 5857)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 5857)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 30, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: LgBiT-KRAS (WT)_SmBiT-CRAF-RBD Bidirectional V...
FEATURES             Location/Qualifiers
     source          1..5857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(747..1541)
                     /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(858..877)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1468..1487
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1573..1620)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     protein_bind    1630..1663
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    1778..1899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1869..1888
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2399..2418)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2400..2418)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2460..2479)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          2462..2594
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        complement(2730..2933)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2759..2779)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2934..3237
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3357..3375
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3357..3374
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             3945..4511
                     /codon_start=1
                     /product="human small GTPase proto-oncoprotein"
                     /label=K-Ras
                     /translation="MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVK
                     DSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREI
                     RKHKEKMSKDGKKKKKKSKTKCVIM"
     polyA_signal    4714..4795
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4874..5231
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4874..5065
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4897..4917)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5082..5217
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      5256..5265
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5262..5660
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5684..5732
                     /note="synthetic polyadenylation signal"
ORIGIN
        1 ttgaaatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccggttg
      721 ggcgtcgctt ggtcggtcat ttcgaatcag aagaactcgt caagaaggcg atagaaggcg
      781 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
      841 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
      901 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
      961 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     1021 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     1081 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     1141 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     1201 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     1261 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     1321 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     1381 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     1441 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     1501 gcggccggag aacctgcgtg caatccatct tgttcaatca tgatctgcct cctgggttta
     1561 gttcctcacc ttgtcgtatt atactatgcc gatatactat gccgatgatt aattgtcaac
     1621 agtggatcca taacttcgta taatgtatgc tatacgaagt tatattagaa ttcttaatta
     1681 agattttacc acatttgtag aggttttact tgcttaaaaa aacctcccac atctccccct
     1741 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     1801 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     1861 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgct cgaagcggcc
     1921 ttgctcagcg gtggcagcag ccaactcagc ttcctttcgg gctttgttag cagccggatc
     1981 agcttgcatg cctgcaggtc gactctagag gatccccggg taccgagccc gaattcgttt
     2041 aaacatgatc caggaaatct acttgaagtt cttctccaat caaagacgca gcatcagtat
     2101 tccaatctaa gcgtgctttt ttacctttgt gttcgtggag aagtctgaac actgcacagc
     2161 actctggttg caggcccctc accttgagtg ctttcataag gcagtcatgc aagctcattc
     2221 catttcgcac attgaccact gttctttgct tgttcggcaa gaaaacacgg atagtgttgc
     2281 ttgtcttaga aggcatggcg atcgcaccgc tcgagcctcc acctccgctc ccgccaccac
     2341 cggagctccc gagaatctcc tcgaacagcc ggtagccggt caccatggtg agcgctagcc
     2401 ctatagtgag tcgtattaat actctagcct taagagctgt aattgaactg ggagtggaca
     2461 cctgtggaga gaaaggcaaa gtggatgtca gtaagaccaa taggtgccta tcagaaacgc
     2521 aagagtcttc tctgtctcga caagcccagt ttctattggt ctccttaaac ctgtcttgta
     2581 accttgatac ttacctgccc agtgcctcac gaccaacttc tgcagcttaa gttcgagact
     2641 gttgtgtcag aagcactgac tgcgttagca atttaactgt gataaactac cgcaataaag
     2701 cttctagtga tctgacggtt cactaaacca gctctgctta tatagacctc ccaccgtaca
     2761 cgcctaccgc ccatttgcgt caacggggcg gggttattac gacattttgg aaagtcccgt
     2821 tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc
     2881 cgtgagtcaa accgctatcc acgcccattg gtgtactgcc aaaaccgcat caccatggta
     2941 atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccgtaa ggtcatgtac
     3001 tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg cggacttggc
     3061 atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga
     3121 cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat
     3181 gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg
     3241 aactccatat atgggctatg aactaatgac cccgtaattg attactatta ataactagtg
     3301 gcgcgccagg gaggcctata taagcagagc tggtttagtg aaccgtcaga tcgcccattt
     3361 aggtgacact atagaataca agctacttgt tctttttgca gctagcgctc accatggtct
     3421 tcacactcga agatttcgtt ggggactggg aacagacagc cgcctacaac ctggaccaag
     3481 tccttgaaca gggaggtgtg tccagtttgc tgcagaatct cgccgtgtcc gtaactccga
     3541 tccaaaggat tgtccggagc ggtgaaaatg ccctgaagat cgacatccat gtcatcatcc
     3601 cgtatgaagg tctgagcgcc gaccaaatgg cccagatcga agaggtgttt aaggtggtgt
     3661 accctgtgga tgatcatcac tttaaggtga tcctgcccta tggcacactg gtaatcgacg
     3721 gggttacgcc gaacatgctg aactatttcg gacggccgta tgaaggcatc gccgtgttcg
     3781 acggcaaaaa gatcactgta acagggaccc tgtggaacgg caacaaaatt atcgacgagc
     3841 gcctgatcac ccccgacggc tccatgctgt tccgagtaac catcaacagt gggagctccg
     3901 gtggtggcgg gagcggaggt ggaggctcga gcggtgcgat cgccatgact gaatataaac
     3961 ttgtggtagt tggagctggt ggcgtaggca agagtgcctt gacgatacag ctaattcaga
     4021 atcattttgt ggacgaatat gatccaacaa tagaggattc ctacaggaag caagtagtaa
     4081 ttgatggaga aacctgtctc ttggatattc tcgacacagc aggtcaagag gagtacagtg
     4141 caatgaggga ccagtacatg aggactgggg agggctttct ttgtgtattt gccataaata
     4201 atactaaatc atttgaagat attcaccatt atagagaaca aattaaaaga gttaaggact
     4261 ctgaagatgt acctatggtc ctagtaggaa ataaatgtga tttgccttct agaacagtag
     4321 acacaaaaca ggctcaggac ttagcaagaa gttatggaat tccttttatt gaaacatcag
     4381 caaagacaag acagggtgtt gatgatgcct tctatacatt agttcgagaa attcgaaaac
     4441 ataaagaaaa gatgagcaaa gatggtaaaa agaagaaaaa gaagtcaaag acaaagtgtg
     4501 taattatgta agtttaaacg aattcgggct cggtacccgg ggatcctcta gaaacaccta
     4561 ggctctcgta cgaagtcagc tgataacgtc gacgtattgc ggccgcggat cataatcagc
     4621 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     4681 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     4741 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgccgacg
     4801 tcttaattaa cgcgtcttgc gcagcaccat ggcctgaaat aacctctgaa agaggaactt
     4861 ggttaggtac cttctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     4921 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4981 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     5041 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     5101 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     5161 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     5221 gcttttgcaa aaagctcgat tcttctgaca ctagcgccac catggccaag cctttgtctc
     5281 aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg
     5341 aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca
     5401 atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg
     5461 ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct
     5521 tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca
     5581 tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg
     5641 gttatgtgtg ggagggctaa actctagcca tcacgatggc cgcaataaaa tatctttatt
     5701 ttcattacat ctgtgtgttg gttttttgtg tgacgattcc ggactataaa aggatctagg
     5761 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5821 gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
