LOCUS       sequence-479920-        6246 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express CRAF-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 6246)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6246)
  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            "Unpublished"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/CRAF-SmBiT/BlastR Vector
FEATURES             Location/Qualifiers
     source          1..6246
                     /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"
     polyA_signal    3309..3430
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3320..3339)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3545..3578)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3588..3635
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3667..4461
                     /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(3721..3740)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4331..4350
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4619..5207)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4699..4718)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5333..5381
                     /note="synthetic polyadenylation signal"
     CDS             complement(5405..5803)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5800..5809
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5834..6191)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5848..5983
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6000..6191
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6148..6168
                     /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 ggagcacata
     1201 cagggagctt ggaagacgat cagcaatggt tttggattca aagatgccgt gtttgatggc
     1261 tccagctgca tctctcctac aatagttcag cagtttggct atcagcgccg ggcatcagat
     1321 gatggcaaac tcacagatcc ttctaagaca agcaacacta tccgtgtttt cttgccgaac
     1381 aagcaaagaa cagtggtcaa tgtgcgaaat ggaatgagct tgcatgactg ccttatgaaa
     1441 gcactcaagg tgaggggcct gcaaccagag tgctgtgcag tgttcagact tctccacgaa
     1501 cacaaaggta aaaaagcacg cttagattgg aatactgatg ctgcgtcttt gattggagaa
     1561 gaacttcaag tagatttcct ggatcatgtt cccctcacaa cacacaactt tgctcggaag
     1621 acgttcctga agcttgcctt ctgtgacatc tgtcagaaat tcctgctcaa tggatttcga
     1681 tgtcagactt gtggctacaa atttcatgag cactgtagca ccaaagtacc tactatgtgt
     1741 gtggactgga gtaacatcag acaactctta ttgtttccaa attccactat tggtgatagt
     1801 ggagtcccag cactaccttc tttgactatg cgtcgtatgc gagagtctgt ttccaggatg
     1861 cctgttagtt ctcagcacag atattctaca cctcacgcct tcacctttaa cacctccagt
     1921 ccctcatctg aaggttccct ctcccagagg cagaggtcga catccacacc taatgtccac
     1981 atggtcagca ccaccctgcc tgtggacagc aggatgattg aggatgcaat tcgaagtcac
     2041 agcgaatcag cctcaccttc agccctgtcc agtagcccca acaatctgag cccaacaggc
     2101 tggtcacagc cgaaaacccc cgtgccagca caaagagagc gggcaccagt atctgggacc
     2161 caggagaaaa acaaaattag gcctcgtgga cagagagatt caagctatta ttgggaaata
     2221 gaagccagtg aagtgatgct gtccactcgg attgggtcag gctcttttgg aactgtttat
     2281 aagggtaaat ggcacggaga tgttgcagta aagatcctaa aggttgtcga cccaacccca
     2341 gagcaattcc aggccttcag gaatgaggtg gctgttctgc gcaaaacacg gcatgtgaac
     2401 attctgcttt tcatggggta catgacaaag gacaacctgg caattgtgac ccagtggtgc
     2461 gagggcagca gcctctacaa acacctgcat gtccaggaga ccaagtttca gatgttccag
     2521 ctaattgaca ttgcccggca gacggctcag ggaatggact atttgcatgc aaagaacatc
     2581 atccatagag acatgaaatc caacaatata tttctccatg aaggcttaac agtgaaaatt
     2641 ggagattttg gtttggcaac agtaaagtca cgctggagtg gttctcagca ggttgaacaa
     2701 cctactggct ctgtcctctg gatggcccca gaggtgatcc gaatgcagga taacaaccca
     2761 ttcagtttcc agtcggatgt ctactcctat ggcatcgtat tgtatgaact gatgacgggg
     2821 gagcttcctt attctcacat caacaaccga gatcagatca tcttcatggt gggccgagga
     2881 tatgcctccc cagatcttag taagctatat aagaactgcc ccaaagcaat gaagaggctg
     2941 gtagctgact gtgtgaagaa agtaaaggaa gagaggcctc tttttcccca gatcctgtct
     3001 tccattgagc tgctccaaca ctctctaccg aagatcaacc ggagcgcttc cgagccatcc
     3061 ttgcatcggg cagcccacac tgaggatatc aatgcttgca cgctgaccac gtccccgagg
     3121 ctgcctgtct tcgtttctca aggcagttca ggtggtggcg ggagcggagg tggaggctcg
     3181 agcggtgtga ccggctaccg gctgttcgag gagattctgt aaggccgcga ctctagagtc
     3241 gacctgcagg catgcaagct gatttggctg ctgccaccgc tgagcaaggc cgcttcgagc
     3301 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     3361 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     3421 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg
     3481 ggaggttttt ttaagcaagt aaaacctcta caaatgtggt aaaatcttaa ttaagaattc
     3541 taatataact tcgtatagca tacattatac gaagttatgg atccactgtt gacaattaat
     3601 catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa acccaggagg
     3661 cagatcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     3721 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     3781 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     3841 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     3901 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     3961 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4021 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4081 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4141 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     4201 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4261 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4321 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4381 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4441 cttcttgacg agttcttctg attcgaaatg accgaccaag cgacgcccaa ccggtatcag
     4501 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4561 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4621 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4681 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4741 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4801 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4861 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4921 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4981 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5041 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5101 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5161 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atttcaagaa gatcctttga
     5221 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5281 tgagattatc aaaaaggatc ttcacctaga tccttttata gtccggaatc gtcacacaaa
     5341 aaaccaacac acagatgtaa tgaaaataaa gatattttat tgcggccatc gtgatggcta
     5401 gagtttagcc ctcccacaca taaccagagg gcagcaattc acgaatccca actgccgtcg
     5461 gctgtccatc actgtccttc actatggctt tgatcccagg atgcagatcg agaagcacct
     5521 gtcggcaccg tccgcagggg ctcaagatgc ccctgttctc atttccgatc gcgacgatac
     5581 aagtcaggtt gccagctgcc gcagcagcag cagtgcccag caccacgagt tctgcacaag
     5641 gtcccccagt aaaatgatat acattgacac cagtgaagat gcggccgtcg ctagagagag
     5701 ctgcgctggc gacgctgtag tcttcagaga tggggatgct gttgattgta gccgttgctc
     5761 tttcaatgag ggtggattct tcttgagaca aaggcttggc catggtggcg ctagtgtcag
     5821 aagaatcgag ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga
     5881 atagctcaga ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat
     5941 ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg
     6001 ggactatggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc
     6061 ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc
     6121 ctgctgggga gcctggggac tttccacacc ctaactgaca cacattccac agctggttct
     6181 ttccgcctca gaaggtacct aaccaagttc ctctttcaga ggttatttca ggccatggtg
     6241 ctgcgc
//
