LOCUS       sequence-479917-        6272 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express SmBiT-CRAF 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 6272)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6272)
  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            "Unpublished"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: SmBiT-CRAF BiBiT-Ready Vector
FEATURES             Location/Qualifiers
     source          1..6272
                     /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    3335..3456
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3346..3365)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3571..3604)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3614..3661
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3693..4487
                     /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(3747..3766)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4357..4376
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4645..5233)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4725..4744)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5359..5407
                     /note="synthetic polyadenylation signal"
     CDS             complement(5431..5829)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5826..5835
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5860..6217)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5874..6009
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6026..6217
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6174..6194
                     /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 cgctcaccat ggtgaccggc
     1201 taccggctgt tcgaggagat tctcgggagc tccggtggtg gcgggagcgg aggtggaggc
     1261 tcgagcggtg cgatcgccat ggagcacata cagggagctt ggaagacgat cagcaatggt
     1321 tttggattca aagatgccgt gtttgatggc tccagctgca tctctcctac aatagttcag
     1381 cagtttggct atcagcgccg ggcatcagat gatggcaaac tcacagatcc ttctaagaca
     1441 agcaacacta tccgtgtttt cttgccgaac aagcaaagaa cagtggtcaa tgtgcgaaat
     1501 ggaatgagct tgcatgactg ccttatgaaa gcactcaagg tgaggggcct gcaaccagag
     1561 tgctgtgcag tgttcagact tctccacgaa cacaaaggta aaaaagcacg cttagattgg
     1621 aatactgatg ctgcgtcttt gattggagaa gaacttcaag tagatttcct ggatcatgtt
     1681 cccctcacaa cacacaactt tgctcggaag acgttcctga agcttgcctt ctgtgacatc
     1741 tgtcagaaat tcctgctcaa tggatttcga tgtcagactt gtggctacaa atttcatgag
     1801 cactgtagca ccaaagtacc tactatgtgt gtggactgga gtaacatcag acaactctta
     1861 ttgtttccaa attccactat tggtgatagt ggagtcccag cactaccttc tttgactatg
     1921 cgtcgtatgc gagagtctgt ttccaggatg cctgttagtt ctcagcacag atattctaca
     1981 cctcacgcct tcacctttaa cacctccagt ccctcatctg aaggttccct ctcccagagg
     2041 cagaggtcga catccacacc taatgtccac atggtcagca ccaccctgcc tgtggacagc
     2101 aggatgattg aggatgcaat tcgaagtcac agcgaatcag cctcaccttc agccctgtcc
     2161 agtagcccca acaatctgag cccaacaggc tggtcacagc cgaaaacccc cgtgccagca
     2221 caaagagagc gggcaccagt atctgggacc caggagaaaa acaaaattag gcctcgtgga
     2281 cagagagatt caagctatta ttgggaaata gaagccagtg aagtgatgct gtccactcgg
     2341 attgggtcag gctcttttgg aactgtttat aagggtaaat ggcacggaga tgttgcagta
     2401 aagatcctaa aggttgtcga cccaacccca gagcaattcc aggccttcag gaatgaggtg
     2461 gctgttctgc gcaaaacacg gcatgtgaac attctgcttt tcatggggta catgacaaag
     2521 gacaacctgg caattgtgac ccagtggtgc gagggcagca gcctctacaa acacctgcat
     2581 gtccaggaga ccaagtttca gatgttccag ctaattgaca ttgcccggca gacggctcag
     2641 ggaatggact atttgcatgc aaagaacatc atccatagag acatgaaatc caacaatata
     2701 tttctccatg aaggcttaac agtgaaaatt ggagattttg gtttggcaac agtaaagtca
     2761 cgctggagtg gttctcagca ggttgaacaa cctactggct ctgtcctctg gatggcccca
     2821 gaggtgatcc gaatgcagga taacaaccca ttcagtttcc agtcggatgt ctactcctat
     2881 ggcatcgtat tgtatgaact gatgacgggg gagcttcctt attctcacat caacaaccga
     2941 gatcagatca tcttcatggt gggccgagga tatgcctccc cagatcttag taagctatat
     3001 aagaactgcc ccaaagcaat gaagaggctg gtagctgact gtgtgaagaa agtaaaggaa
     3061 gagaggcctc tttttcccca gatcctgtct tccattgagc tgctccaaca ctctctaccg
     3121 aagatcaacc ggagcgcttc cgagccatcc ttgcatcggg cagcccacac tgaggatatc
     3181 aatgcttgca cgctgaccac gtccccgagg ctgcctgtct tcgtttaaac gaattcgggc
     3241 tcggtacccg gggatcctct agagtcgacc tgcaggcatg caagctgatt tggctgctgc
     3301 caccgctgag caaggccgct tcgagcagac atgataagat acattgatga gtttggacaa
     3361 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     3421 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     3481 atgtttcagg ttcaggggga gatgtgggag gtttttttaa gcaagtaaaa cctctacaaa
     3541 tgtggtaaaa tcttaattaa gaattctaat ataacttcgt atagcataca ttatacgaag
     3601 ttatggatcc actgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
     3661 caaggtgagg aactaaaccc aggaggcaga tcatgattga acaagatgga ttgcacgcag
     3721 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3781 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3841 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     3901 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3961 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     4021 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     4081 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     4141 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     4201 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4261 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     4321 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     4381 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4441 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgattc gaaatgaccg
     4501 accaagcgac gcccaaccgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4561 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4621 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4681 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4741 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4801 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4861 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4921 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4981 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5041 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     5101 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5161 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5221 aaaaggattt caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5281 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5341 tttatagtcc ggaatcgtca cacaaaaaac caacacacag atgtaatgaa aataaagata
     5401 ttttattgcg gccatcgtga tggctagagt ttagccctcc cacacataac cagagggcag
     5461 caattcacga atcccaactg ccgtcggctg tccatcactg tccttcacta tggctttgat
     5521 cccaggatgc agatcgagaa gcacctgtcg gcaccgtccg caggggctca agatgcccct
     5581 gttctcattt ccgatcgcga cgatacaagt caggttgcca gctgccgcag cagcagcagt
     5641 gcccagcacc acgagttctg cacaaggtcc cccagtaaaa tgatatacat tgacaccagt
     5701 gaagatgcgg ccgtcgctag agagagctgc gctggcgacg ctgtagtctt cagagatggg
     5761 gatgctgttg attgtagccg ttgctctttc aatgagggtg gattcttctt gagacaaagg
     5821 cttggccatg gtggcgctag tgtcagaaga atcgagcttt ttgcaaaagc ctaggcctcc
     5881 aaaaaagcct cctcactact tctggaatag ctcagaggcc gaggcggcct cggcctctgc
     5941 ataaataaaa aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag
     6001 gggcgggatg ggcggagtta ggggcgggac tatggttgct gactaattga gatgcatgct
     6061 ttgcatactt ctgcctgctg gggagcctgg ggactttcca cacctggttg ctgactaatt
     6121 gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc cacaccctaa
     6181 ctgacacaca ttccacagct ggttctttcc gcctcagaag gtacctaacc aagttcctct
     6241 ttcagaggtt atttcaggcc atggtgctgc gc
//
