LOCUS       sequence-487373-        7984 bp    DNA     circular SYN 29-SEP-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 7984)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7984)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 29, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: CRAF-SmBiT, BiBiT-Ready Vector
FEATURES             Location/Qualifiers
     source          1..7984
                     /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(4017..4036)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4018..4036)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4078..4097)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          4080..4212
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        complement(4348..4551)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4377..4397)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4552..4855
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    5088..5169
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5193..6371
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5424..6362
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     6319..6339
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             6750..7787
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             7766..7774
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    7811..7859
                     /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 ccaaatcagc ttgcatgcct gcaggtcgac tctagagtcg
     1981 cggccttaca gaatctcctc gaacagccgg tagccggtca cggagccccc tccacctgag
     2041 ccacctcctc cgagagaaac gaagacaggc agcctcgggg acgtggtcag cgtgcaagca
     2101 ttgatatcct cagtgtgggc tgcccgatgc aaggatggct cggaagcgct ccggttgatc
     2161 ttcggtagag agtgttggag cagctcaatg gaagacagga tctggggaaa aagaggcctc
     2221 tcttccttta ctttcttcac acagtcagct accagcctct tcattgcttt ggggcagttc
     2281 ttatatagct tactaagatc tggggaggca tatcctcggc ccaccatgaa gatgatctga
     2341 tctcggttgt tgatgtgaga ataaggaagc tcccccgtca tcagttcata caatacgatg
     2401 ccataggagt agacatccga ctggaaactg aatgggttgt tatcctgcat tcggatcacc
     2461 tctggggcca tccagaggac agagccagta ggttgttcaa cctgctgaga accactccag
     2521 cgtgacttta ctgttgccaa accaaaatct ccaattttca ctgttaagcc ttcatggaga
     2581 aatatattgt tggatttcat gtctctatgg atgatgttct ttgcatgcaa atagtccatt
     2641 ccctgagccg tctgccgggc aatgtcaatt agctggaaca tctgaaactt ggtctcctgg
     2701 acatgcaggt gtttgtagag gctgctgccc tcgcaccact gggtcacaat tgccaggttg
     2761 tcctttgtca tgtaccccat gaaaagcaga atgttcacat gccgtgtttt gcgcagaaca
     2821 gccacctcat tcctgaaggc ctggaattgc tctggggttg ggtcgacaac ctttaggatc
     2881 tttactgcaa catctccgtg ccatttaccc ttataaacag ttccaaaaga gcctgaccca
     2941 atccgagtgg acagcatcac ttcactggct tctatttccc aataatagct tgaatctctc
     3001 tgtccacgag gcctaatttt gtttttctcc tgggtcccag atactggtgc ccgctctctt
     3061 tgtgctggca cgggggtttt cggctgtgac cagcctgttg ggctcagatt gttggggcta
     3121 ctggacaggg ctgaaggtga ggctgattcg ctgtgacttc gaattgcatc ctcaatcatc
     3181 ctgctgtcca caggcagggt ggtgctgacc atgtggacat taggtgtgga tgtcgacctc
     3241 tgcctctggg agagggaacc ttcagatgag ggactggagg tgttaaaggt gaaggcgtga
     3301 ggtgtagaat atctgtgctg agaactaaca ggcatcctgg aaacagactc tcgcatacga
     3361 cgcatagtca aagaaggtag tgctgggact ccactatcac caatagtgga atttggaaac
     3421 aataagagtt gtctgatgtt actccagtcc acacacatag taggtacttt ggtgctacag
     3481 tgctcatgaa atttgtagcc acaagtctga catcgaaatc cattgagcag gaatttctga
     3541 cagatgtcac agaaggcaag cttcaggaac gtcttccgag caaagttgtg tgttgtgagg
     3601 ggaacatgat ccaggaaatc tacttgaagt tcttctccaa tcaaagacgc agcatcagta
     3661 ttccaatcta agcgtgcttt tttacctttg tgttcgtgga gaagtctgaa cactgcacag
     3721 cactctggtt gcaggcccct caccttgagt gctttcataa ggcagtcatg caagctcatt
     3781 ccatttcgca cattgaccac tgttctttgc ttgttcggca agaaaacacg gatagtgttg
     3841 cttgtcttag aaggatctgt gagtttgcca tcatctgatg cccggcgctg atagccaaac
     3901 tgctgaacta ttgtaggaga gatgcagctg gagccatcaa acacggcatc tttgaatcca
     3961 aaaccattgc tgatcgtctt ccaagctccc tgtatgtgct ccatggcgat cgctagccct
     4021 atagtgagtc gtattaatac tctagcctta agagctgtaa ttgaactggg agtggacacc
     4081 tgtggagaga aaggcaaagt ggatgtcagt aagaccaata ggtgcctatc agaaacgcaa
     4141 gagtcttctc tgtctcgaca agcccagttt ctattggtct ccttaaacct gtcttgtaac
     4201 cttgatactt acctgcccag tgcctcacga ccaacttctg cagcttaagt tcgagactgt
     4261 tgtgtcagaa gcactgactg cgttagcaat ttaactgtga taaactaccg caataaagct
     4321 tctagtgatc tgacggttca ctaaaccagc tctgcttata tagacctccc accgtacacg
     4381 cctaccgccc atttgcgtca acggggcggg gttattacga cattttggaa agtcccgttg
     4441 attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg gaaatccccg
     4501 tgagtcaaac cgctatccac gcccattggt gtactgccaa aaccgcatca ccatggtaat
     4561 agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccgtaagg tcatgtactg
     4621 ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg gacttggcat
     4681 atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg
     4741 tcaatggaaa gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg
     4801 gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa
     4861 ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat aactagtggc
     4921 gcgccataat cctaggctct cgtacgaagt cagctgataa cgtcgacgta ttgcggccgc
     4981 ggatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     5041 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     5101 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5161 tttcactgcc gacgtcttaa ttaacgcgtc ttggctccgg tgcccgtcag tgggcagagc
     5221 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct
     5281 agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
     5341 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
     5401 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct
     5461 ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctgcag tacgtgattc
     5521 ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag
     5581 ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa
     5641 tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt
     5701 tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag
     5761 atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc
     5821 agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt
     5881 agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc
     5941 cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc
     6001 ccggccctgc tgcaggcagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg
     6061 agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca
     6121 cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg agtacgtcgt
     6181 ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag tgggtggaga
     6241 ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt
     6301 ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc
     6361 aggtgtcgtg aaagccgaat tctgcagata tccatcacac tgtcgagcat gcatctagtc
     6421 cagtgtggtg gaattcggct tcgtggggac gccgtacagg gacgtgcacc tctcccgctg
     6481 caccgcctcc agcgtcgccg ccggctcgaa ggacggggcc gggatgacga tgcaggcggc
     6541 gtgggaggtg gcgcccaagt tgcccatgac catgccgaag cagtggtaga agggcaccgg
     6601 cagacacacc cggtcctgct ccgtgtagcc gaccgtgcgg cccacccagt agccgttgtt
     6661 gaggatgttg tggtgggaga gcgtggcgcc cttggggaag ccggtggtgc cggaggtgta
     6721 ctggatgttg accgggaatc gatgccacca tgaagaagcc cgaactcacc gctaccagcg
     6781 ttgaaaaatt tctcatcgag aagttcgaca gtgtgagcga cctgatgcag ttgtcggagg
     6841 gcgaagagag ccgagccttc agcttcgatg tcggcggacg cggctatgta ctgcgggtga
     6901 atagctgcgc tgatggcttc tacaaagacc gctacgtgta ccgccacttc gccagcgctg
     6961 cactacccat ccccgaagtg ttggacatcg gcgagttcag cgagagcctg acatactgca
     7021 tcagtagacg cgcccaaggc gttactctcc aagacctccc cgaaacagag ctgcctgctg
     7081 tgttacagcc tgtcgccgaa gctatggatg ctattgccgc cgccgacctc agtcaaacca
     7141 gcggcttcgg cccattcggg ccccaaggca tcggccagta cacaacctgg cgggatttca
     7201 tttgcgccat tgctgatccc catgtctacc actggcagac cgtgatggac gacaccgtgt
     7261 ccgccagcgt agctcaagcc ctggacgaac tgatgctgtg ggccgaagac tgtcccgagg
     7321 tgcgccacct cgtccatgcc gacttcggca gcaacaacgt cctgaccgac aacggccgca
     7381 tcaccgccgt aatcgactgg tccgaagcta tgttcgggga cagtcagtac gaggtggcca
     7441 acatcttctt ctggcggccc tggctggctt gcatggagca gcagactcgc tacttcgagc
     7501 gccggcatcc cgagctggcc ggcagccctc gtctgcgagc ctacatgctg cgcatcggcc
     7561 tggatcagct ctaccagagc ctcgtggacg gcaacttcga cgatgctgcc tgggctcaag
     7621 gccgctgcga tgccatcgtc cgcagcgggg ccggcaccgt cggtcgcaca caaatcgctc
     7681 gccggagcgc agccgtatgg accgacggct gcgtcgaggt gctggccgac agcggcaacc
     7741 gccggcccag tacacgaccg cgcgctaagg aggtaggtcg agtttagact ctagccatca
     7801 cgatggccgc aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga
     7861 cgattccgga ctataaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     7921 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7981 cttc
//
