LOCUS       sequence-479898-        7932 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Express EGFR-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 7932)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7932)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: EGFR-SmBiT, BiBiT-Ready Vector
FEATURES             Location/Qualifiers
     source          1..7932
                     /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"
     CDS             join(1189..1260,1261..3123,3124..3192,3193..4818)
                     /codon_start=1
                     /product="precursor of human epidermal growth factor 
                     receptor"
                     /label=EGFR
                     /translation="MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFE
                     DHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENL
                     QIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQ
                     WRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGR
                     CRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNP
                     EGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGI
                     GIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVK
                     EITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISD
                     GDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWG
                     PEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGP
                     DNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEG
                     CPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTP
                     SGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSP
                     KANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQ
                     YLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHA
                     EGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKG
                     ERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHL
                     PSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVA
                     CIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQN
                     PVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQIS
                     LDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA"
     polyA_signal    4995..5116
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5006..5025)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(5231..5264)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5274..5321
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5353..6147
                     /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(5407..5426)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6017..6036
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(6305..6893)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6385..6404)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    7019..7067
                     /note="synthetic polyadenylation signal"
     CDS             complement(7091..7489)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      7486..7495
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7520..7877)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7534..7669
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        7686..7877
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     7834..7854
                     /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 gcgaccctcc
     1201 gggacggccg gggcagcgct cctggcgctg ctggctgcgc tctgcccggc gagtcgggct
     1261 ctggaggaaa agaaagtttg ccaaggcacg agtaacaagc tcacgcagtt gggcactttt
     1321 gaagatcatt ttctcagcct ccagaggatg ttcaataact gtgaggtggt ccttgggaat
     1381 ttggaaatta cctatgtgca gaggaattat gatctttcct tcttaaagac catccaggag
     1441 gtggctggtt atgtcctcat tgccctcaac acagtggagc gaattccttt ggaaaacctg
     1501 cagatcatca gaggaaatat gtactacgaa aattcctatg ccttagcagt cttatctaac
     1561 tatgatgcaa ataaaaccgg actgaaggag ctgcccatga gaaatttaca ggaaatcctg
     1621 catggcgccg tgcggttcag caacaaccct gccctgtgca acgtggagag catccagtgg
     1681 cgggacatag tcagcagtga ctttctcagc aacatgtcga tggacttcca gaaccacctg
     1741 ggcagctgcc aaaagtgtga tccaagctgt cccaatggga gctgctgggg tgcaggagag
     1801 gagaactgcc agaaactgac caaaatcatc tgtgcccagc agtgctccgg gcgctgccgt
     1861 ggcaagtccc ccagtgactg ctgccacaac cagtgtgctg caggctgcac aggcccccgg
     1921 gagagcgact gcctggtctg ccgcaaattc cgagacgaag ccacgtgcaa ggacacctgc
     1981 cccccactca tgctctacaa ccccaccacg taccagatgg atgtgaaccc cgagggcaaa
     2041 tacagctttg gtgccacctg cgtgaagaag tgtccccgta attatgtggt gacagatcac
     2101 ggctcgtgcg tccgagcctg tggggccgac agctatgaga tggaggaaga cggcgtccgc
     2161 aagtgtaaga agtgcgaagg gccttgccgc aaagtgtgta acggaatagg tattggtgaa
     2221 tttaaagact cactctccat aaatgctacg aatattaaac acttcaaaaa ctgcacctcc
     2281 atcagtggcg atctccacat cctgccggtg gcatttaggg gtgactcctt cacacatact
     2341 cctcctctgg atccacagga actggatatt ctgaaaaccg taaaggaaat cacagggttt
     2401 ttgctgattc aggcttggcc tgaaaacagg acggacctcc atgcctttga gaacctagaa
     2461 atcatacgcg gcaggaccaa gcaacatggt cagttttctc ttgcagtcgt cagcctgaac
     2521 ataacatcct tgggattacg ctccctcaag gagataagtg atggagatgt gataatttca
     2581 ggaaacaaaa atttgtgcta tgcaaataca ataaactgga aaaaactgtt tgggacctcc
     2641 ggtcagaaaa ccaaaattat aagcaacaga ggtgaaaaca gctgcaaggc cacaggccag
     2701 gtctgccatg ccttgtgctc ccccgagggc tgctggggcc cggagcccag ggactgcgtc
     2761 tcttgccgga atgtcagccg aggcagggaa tgcgtggaca agtgcaacct tctggagggt
     2821 gagccaaggg agtttgtgga gaactctgag tgcatacagt gccacccaga gtgcctgcct
     2881 caggccatga acatcacctg cacaggacgg ggaccagaca actgtatcca gtgtgcccac
     2941 tacattgacg gcccccactg cgtcaagacc tgcccggcag gagtcatggg agaaaacaac
     3001 accctggtct ggaagtacgc agacgccggc catgtgtgcc acctgtgcca tccaaactgc
     3061 acctacggat gcacagggcc aggtcttgaa ggctgtccaa cgaatgggcc taagatcccg
     3121 tccatcgcca ctgggatggt gggggccctc ctcttgctgc tggtggtggc cctggggatc
     3181 ggcctcttca tgcgaaggcg ccacatcgtt cggaagcgca cgctgcggag gctgctgcag
     3241 gagagggagc ttgtggagcc tcttacaccc agtggagaag ctcccaacca agctctcttg
     3301 aggatcttga aggaaactga attcaaaaag atcaaagtgc tgggctccgg tgcgttcggc
     3361 acggtgtata agggactctg gatcccagaa ggtgagaaag ttaaaattcc cgtcgctatc
     3421 aaggaattaa gagaagcaac atctccgaaa gccaacaagg aaatcctcga tgaagcctac
     3481 gtgatggcca gcgtggacaa cccccacgtg tgccgcctgc tgggcatctg cctcacctcc
     3541 accgtgcagc tcatcacgca gctcatgccc ttcggctgcc tcctggacta tgtccgggaa
     3601 cacaaagaca atattggctc ccagtacctg ctcaactggt gtgtgcagat cgcaaagggc
     3661 atgaactact tggaggaccg tcgcttggtg caccgcgacc tggcagccag gaacgtactg
     3721 gtgaaaacac cgcagcatgt caagatcaca gattttgggc tggccaaact gctgggtgcg
     3781 gaagagaaag aataccatgc agaaggaggc aaagtgccta tcaagtggat ggcattggaa
     3841 tcaattttac acagaatcta tacccaccag agtgatgtct ggagctacgg ggtgaccgtt
     3901 tgggagttga tgacctttgg atccaagcca tatgacggaa tccctgccag cgagatctcc
     3961 tccatcctgg agaaaggaga acgcctccct cagccaccca tatgtaccat cgatgtctac
     4021 atgatcatgg tcaagtgctg gatgatagac gcagatagtc gcccaaagtt ccgtgagttg
     4081 atcatcgaat tctccaaaat ggcccgagac ccccagcgct accttgtcat tcagggggat
     4141 gaaagaatgc atttgccaag tcctacagac tccaacttct accgtgccct gatggatgaa
     4201 gaagacatgg acgacgtggt ggatgccgac gagtacctca tcccacagca gggcttcttc
     4261 agcagcccct ccacgtcacg gactcccctc ctgagctctc tgagtgcaac cagcaacaat
     4321 tccaccgtgg cttgcattga tagaaatggg ctgcaaagct gtcccatcaa ggaagacagc
     4381 ttcttgcagc gatacagctc agaccccaca ggcgccttga ctgaggacag catagacgac
     4441 accttcctcc cagtgcctga atacataaac cagtccgttc ccaaaaggcc cgctggctct
     4501 gtgcagaatc ctgtctatca caatcagcct ctgaaccccg cgcccagcag agacccacac
     4561 taccaggacc cccacagcac tgcagtgggc aaccccgagt atctcaacac tgtccagccc
     4621 acctgtgtca acagcacatt cgacagccct gcccactggg cccagaaagg cagccaccaa
     4681 attagcctgg acaaccctga ctaccagcag gacttctttc ccaaggaagc caagccaaat
     4741 ggcatcttta agggctccac agctgaaaat gcagaatacc taagggtcgc gccacaaagc
     4801 agtgaattta ttggagcagt ttctcaaggc agttcaggtg gtggcgggag cggaggtgga
     4861 ggctcgagcg gtgtgaccgg ctaccggctg ttcgaggaga ttctgtaagg ccgcgactct
     4921 agagtcgacc tgcaggcatg caagctgatt tggctgctgc caccgctgag caaggccgct
     4981 tcgagcagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     5041 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     5101 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     5161 gatgtgggag gtttttttaa gcaagtaaaa cctctacaaa tgtggtaaaa tcttaattaa
     5221 gaattctaat ataacttcgt atagcataca ttatacgaag ttatggatcc actgttgaca
     5281 attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaaccc
     5341 aggaggcaga tcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     5401 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     5461 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     5521 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     5581 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     5641 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     5701 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     5761 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     5821 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     5881 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     5941 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     6001 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     6061 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     6121 tatcgccttc ttgacgagtt cttctgattc gaaatgaccg accaagcgac gcccaaccgg
     6181 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     6241 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6301 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6361 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6421 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6481 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6541 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6601 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6661 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6721 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     6781 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6841 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggattt caagaagatc
     6901 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     6961 tggtcatgag attatcaaaa aggatcttca cctagatcct tttatagtcc ggaatcgtca
     7021 cacaaaaaac caacacacag atgtaatgaa aataaagata ttttattgcg gccatcgtga
     7081 tggctagagt ttagccctcc cacacataac cagagggcag caattcacga atcccaactg
     7141 ccgtcggctg tccatcactg tccttcacta tggctttgat cccaggatgc agatcgagaa
     7201 gcacctgtcg gcaccgtccg caggggctca agatgcccct gttctcattt ccgatcgcga
     7261 cgatacaagt caggttgcca gctgccgcag cagcagcagt gcccagcacc acgagttctg
     7321 cacaaggtcc cccagtaaaa tgatatacat tgacaccagt gaagatgcgg ccgtcgctag
     7381 agagagctgc gctggcgacg ctgtagtctt cagagatggg gatgctgttg attgtagccg
     7441 ttgctctttc aatgagggtg gattcttctt gagacaaagg cttggccatg gtggcgctag
     7501 tgtcagaaga atcgagcttt ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact
     7561 tctggaatag ctcagaggcc gaggcggcct cggcctctgc ataaataaaa aaaattagtc
     7621 agccatgggg cggagaatgg gcggaactgg gcggagttag gggcgggatg ggcggagtta
     7681 ggggcgggac tatggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg
     7741 gggagcctgg ggactttcca cacctggttg ctgactaatt gagatgcatg ctttgcatac
     7801 ttctgcctgc tggggagcct ggggactttc cacaccctaa ctgacacaca ttccacagct
     7861 ggttctttcc gcctcagaag gtacctaacc aagttcctct ttcagaggtt atttcaggcc
     7921 atggtgctgc gc
//
