LOCUS       sequence-479899-        8991 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express EGFR(1-673) 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 8991)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8991)
  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            "Unpublished"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: NanoBiT EGFR(1-673) Assay, BiBiT vector
FEATURES             Location/Qualifiers
     source          1..8991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(2870..2888)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2871..2888)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        3008..3311
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3312..3515
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3466..3486
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          3651..3783
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     3766..3785
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     3827..3846
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3827..3845
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    6054..6175
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6065..6084)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(6290..6323)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        6333..6380
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6412..7206
                     /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(6466..6485)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7076..7095
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(7364..7952)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7444..7463)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    8078..8126
                     /note="synthetic polyadenylation signal"
     CDS             complement(8150..8548)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      8545..8554
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(8579..8936)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      8593..8728
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        8745..8936
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     8893..8913
                     /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 ggtgtttcta gagtcgcggc cttagctgtt gatggttact
      301 cggaacagca tggagccgtc gggggtgatc aggcgctcgt cgataatttt gttgccgttc
      361 cacagggtcc ctgttacagt gatctttttg ccgtcgaaca cggcgatgcc ttcatacggc
      421 cgtccgaaat agttcagcat gttcggcgta accccgtcga ttaccagtgt gccatagggc
      481 aggatcacct taaagtgatg atcatccaca gggtacacca ccttaaacac ctcttcgatc
      541 tgggccattt ggtcggcgct cagaccttca tacgggatga tgacatggat gtcgatcttc
      601 agggcatttt caccgctccg gacaatcctt tggatcggag ttacggacac ggcgagattc
      661 tgcagcaaac tggacacacc tccctgttca aggacttggt ccaggttgta ggcggctgtc
      721 tgttcccagt ccccaacgaa atcttcgagt gtgaagacac cgctcgagcc tccacctccg
      781 ctcccgccac cacctgaact gccttgagaa acgatgtggc gccttcgcat gaagaggccg
      841 atccccaggg ccaccaccag cagcaagagg agggccccca ccatcccagt ggcgatggac
      901 gggatcttag gcccattcgt tggacagcct tcaagacctg gccctgtgca tccgtaggtg
      961 cagtttggat ggcacaggtg gcacacatgg ccggcgtctg cgtacttcca gaccagggtg
     1021 ttgttttctc ccatgactcc tgccgggcag gtcttgacgc agtgggggcc gtcaatgtag
     1081 tgggcacact ggatacagtt gtctggtccc cgtcctgtgc aggtgatgtt catggcctga
     1141 ggcaggcact ctgggtggca ctgtatgcac tcagagttct ccacaaactc ccttggctca
     1201 ccctccagaa ggttgcactt gtccacgcat tccctgcctc ggctgacatt ccggcaagag
     1261 acgcagtcct tgggctccgg gccccagcag ccctcggggg agcacaaggc atggcagacc
     1321 tggcctgtgg ccttgcagct gttttcacct ctgttgctta taattttggt tttctgaccg
     1381 gaggtcccaa acagtttttt ccagtttatt gtatttgcat agcacaaatt tttgtttcct
     1441 gaaattatca catctccatc acttatctcc ttgagggagc gtaatcccaa ggatgttatg
     1501 ttcaggctga cgactgcaag agaaaactga ccatgttgct tggtcctgcc gcgtatgatt
     1561 tctaggttct caaaggcatg gaggtccgtc ctgttttcag gccaagcctg aatcagcaaa
     1621 aaccctgtga tttcctttac ggttttcaga atatccagtt cctgtggatc cagaggagga
     1681 gtatgtgtga aggagtcacc cctaaatgcc accggcagga tgtggagatc gccactgatg
     1741 gaggtgcagt ttttgaagtg tttaatattc gtagcattta tggagagtga gtctttaaat
     1801 tcaccaatac ctattccgtt acacactttg cggcaaggcc cttcgcactt cttacacttg
     1861 cggacgccgt cttcctccat ctcatagctg tcggccccac aggctcggac gcacgagccg
     1921 tgatctgtca ccacataatt acggggacac ttcttcacgc aggtggcacc aaagctgtat
     1981 ttgccctcgg ggttcacatc catctggtac gtggtggggt tgtagagcat gagtgggggg
     2041 caggtgtcct tgcacgtggc ttcgtctcgg aatttgcggc agaccaggca gtcgctctcc
     2101 cgggggcctg tgcagcctgc agcacactgg ttgtggcagc agtcactggg ggacttgcca
     2161 cggcagcgcc cggagcactg ctgggcacag atgattttgg tcagtttctg gcagttctcc
     2221 tctcctgcac cccagcagct cccattggga cagcttggat cacacttttg gcagctgccc
     2281 aggtggttct ggaagtccat cgacatgttg ctgagaaagt cactgctgac tatgtcccgc
     2341 cactggatgc tctccacgtt gcacagggca gggttgttgc tgaaccgcac ggcgccatgc
     2401 aggatttcct gtaaatttct catgggcagc tccttcagtc cggttttatt tgcatcatag
     2461 ttagataaga ctgctaaggc ataggaattt tcgtagtaca tatttcctct gatgatctgc
     2521 aggttttcca aaggaattcg ctccactgtg ttgagggcaa tgaggacata accagccacc
     2581 tcctggatgg tctttaagaa ggaaagatca taattcctct gcacataggt aatttccaaa
     2641 ttcccaagga ccacctcaca gttattgaac atcctctgga ggctgagaaa atgatcttca
     2701 aaagtgccca actgcgtgag cttgttactc gtgccttggc aaactttctt ttcctccaga
     2761 gcccgactcg ccgggcagag cgcagccagc agcgccagga gcgctgcccc ggccgtcccg
     2821 gagggtcgca tggcgatcgc tagctgcaaa aagaacaagt agcttgtatt ctatagtgtc
     2881 acctaaatgg gcgatctgac ggttcactaa accagctctg cttatatagg cctccctggc
     2941 gcgccactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     3001 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     3061 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     3121 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     3181 atatgccaag tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     3241 cccagtacat gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg
     3301 ctattaccat ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag cggtttgact
     3361 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
     3421 atcaacggga ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa atgggcggta
     3481 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatcacta
     3541 gaagctttat tgcggtagtt tatcacagtt aaattgctaa cgcagtcagt gcttctgaca
     3601 caacagtctc gaacttaagc tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca
     3661 aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga cagagaagac
     3721 tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc tttctctcca
     3781 caggtgtcca ctcccagttc aattacagct cttaaggcta gagtattaat acgactcact
     3841 atagggctag cgatcgccat gcgaccctcc gggacggccg gggcagcgct cctggcgctg
     3901 ctggctgcgc tctgcccggc gagtcgggct ctggaggaaa agaaagtttg ccaaggcacg
     3961 agtaacaagc tcacgcagtt gggcactttt gaagatcatt ttctcagcct ccagaggatg
     4021 ttcaataact gtgaggtggt ccttgggaat ttggaaatta cctatgtgca gaggaattat
     4081 gatctttcct tcttaaagac catccaggag gtggctggtt atgtcctcat tgccctcaac
     4141 acagtggagc gaattccttt ggaaaacctg cagatcatca gaggaaatat gtactacgaa
     4201 aattcctatg ccttagcagt cttatctaac tatgatgcaa ataaaaccgg actgaaggag
     4261 ctgcccatga gaaatttaca ggaaatcctg catggcgccg tgcggttcag caacaaccct
     4321 gccctgtgca acgtggagag catccagtgg cgggacatag tcagcagtga ctttctcagc
     4381 aacatgtcga tggacttcca gaaccacctg ggcagctgcc aaaagtgtga tccaagctgt
     4441 cccaatggga gctgctgggg tgcaggagag gagaactgcc agaaactgac caaaatcatc
     4501 tgtgcccagc agtgctccgg gcgctgccgt ggcaagtccc ccagtgactg ctgccacaac
     4561 cagtgtgctg caggctgcac aggcccccgg gagagcgact gcctggtctg ccgcaaattc
     4621 cgagacgaag ccacgtgcaa ggacacctgc cccccactca tgctctacaa ccccaccacg
     4681 taccagatgg atgtgaaccc cgagggcaaa tacagctttg gtgccacctg cgtgaagaag
     4741 tgtccccgta attatgtggt gacagatcac ggctcgtgcg tccgagcctg tggggccgac
     4801 agctatgaga tggaggaaga cggcgtccgc aagtgtaaga agtgcgaagg gccttgccgc
     4861 aaagtgtgta acggaatagg tattggtgaa tttaaagact cactctccat aaatgctacg
     4921 aatattaaac acttcaaaaa ctgcacctcc atcagtggcg atctccacat cctgccggtg
     4981 gcatttaggg gtgactcctt cacacatact cctcctctgg atccacagga actggatatt
     5041 ctgaaaaccg taaaggaaat cacagggttt ttgctgattc aggcttggcc tgaaaacagg
     5101 acggacctcc atgcctttga gaacctagaa atcatacgcg gcaggaccaa gcaacatggt
     5161 cagttttctc ttgcagtcgt cagcctgaac ataacatcct tgggattacg ctccctcaag
     5221 gagataagtg atggagatgt gataatttca ggaaacaaaa atttgtgcta tgcaaataca
     5281 ataaactgga aaaaactgtt tgggacctcc ggtcagaaaa ccaaaattat aagcaacaga
     5341 ggtgaaaaca gctgcaaggc cacaggccag gtctgccatg ccttgtgctc ccccgagggc
     5401 tgctggggcc cggagcccaa ggactgcgtc tcttgccgga atgtcagccg aggcagggaa
     5461 tgcgtggaca agtgcaacct tctggagggt gagccaaggg agtttgtgga gaactctgag
     5521 tgcatacagt gccacccaga gtgcctgcct caggccatga acatcacctg cacaggacgg
     5581 ggaccagaca actgtatcca gtgtgcccac tacattgacg gcccccactg cgtcaagacc
     5641 tgcccggcag gagtcatggg agaaaacaac accctggtct ggaagtacgc agacgccggc
     5701 catgtgtgcc acctgtgcca tccaaactgc acctacggat gcacagggcc aggtcttgaa
     5761 ggctgtccaa cgaatgggcc taagatcccg tccatcgcca ctgggatggt gggggccctc
     5821 ctcttgctgc tggtggtggc cctggggatc ggcctcttca tgcgaaggcg ccacatcgtt
     5881 tctcaaggca gttcaggtgg tggcgggagc ggaggtggag gctcgagcgg tgtgaccggc
     5941 taccggctgt tcgaggagat tctgtaaggc cgcgactcta gagtcgacct gcaggcatgc
     6001 aagctgattt ggctgctgcc accgctgagc aaggccgctt cgagcagaca tgataagata
     6061 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     6121 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     6181 caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg tttttttaag
     6241 caagtaaaac ctctacaaat gtggtaaaat cttaattaag aattctaata taacttcgta
     6301 tagcatacat tatacgaagt tatggatcca ctgttgacaa ttaatcatcg gcatagtata
     6361 tcggcatagt ataatacgac aaggtgagga actaaaccca ggaggcagat catgattgaa
     6421 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     6481 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     6541 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     6601 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     6661 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     6721 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     6781 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     6841 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     6901 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     6961 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     7021 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     7081 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     7141 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     7201 ttctgattcg aaatgaccga ccaagcgacg cccaaccggt atcagctcac tcaaaggcgg
     7261 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     7321 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     7381 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     7441 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     7501 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     7561 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     7621 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     7681 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     7741 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     7801 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     7861 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     7921 agcagattac gcgcagaaaa aaaggatttc aagaagatcc tttgatcttt tctacggggt
     7981 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     8041 ggatcttcac ctagatcctt ttatagtccg gaatcgtcac acaaaaaacc aacacacaga
     8101 tgtaatgaaa ataaagatat tttattgcgg ccatcgtgat ggctagagtt tagccctccc
     8161 acacataacc agagggcagc aattcacgaa tcccaactgc cgtcggctgt ccatcactgt
     8221 ccttcactat ggctttgatc ccaggatgca gatcgagaag cacctgtcgg caccgtccgc
     8281 aggggctcaa gatgcccctg ttctcatttc cgatcgcgac gatacaagtc aggttgccag
     8341 ctgccgcagc agcagcagtg cccagcacca cgagttctgc acaaggtccc ccagtaaaat
     8401 gatatacatt gacaccagtg aagatgcggc cgtcgctaga gagagctgcg ctggcgacgc
     8461 tgtagtcttc agagatgggg atgctgttga ttgtagccgt tgctctttca atgagggtgg
     8521 attcttcttg agacaaaggc ttggccatgg tggcgctagt gtcagaagaa tcgagctttt
     8581 tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggccg
     8641 aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg
     8701 cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg
     8761 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
     8821 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     8881 gggactttcc acaccctaac tgacacacat tccacagctg gttctttccg cctcagaagg
     8941 tacctaacca agttcctctt tcagaggtta tttcaggcca tggtgctgcg c
//
