LOCUS       sequence-479809-        6760 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express LgBiT-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 6760)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6760)
  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            "Unpublished"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: LgBiT-CRAF BiBiT-Ready Vector
FEATURES             Location/Qualifiers
     source          1..6760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    17..98
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     42..61
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(2814..2832)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2815..2832)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        2952..3255
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3410..3430
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        3463..3510
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3542..4336
                     /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(3596..3615)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4206..4225
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4494..5082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4574..4593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5208..5256
                     /note="synthetic polyadenylation signal"
     CDS             complement(5280..6317)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             5293..5301
                     /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)"
     promoter        complement(6348..6705)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6362..6497
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6514..6705
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6662..6682
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 aagacgcgtg acgtcggcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
       61 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
      121 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
      181 aatgtggtat ggctgattat gatccgcggc cgcaatacgt cgacgttatc agctgacttc
      241 gtacgagagc ctaggtgttt ctagaggatc cccgggtacc gagcccgaat tcgtttaaac
      301 gaagacaggc agcctcgggg acgtggtcag cgtgcaagca ttgatatcct cagtgtgggc
      361 tgcccgatgc aaggatggct cggaagcgct ccggttgatc ttcggtagag agtgttggag
      421 cagctcaatg gaagacagga tctggggaaa aagaggcctc tcttccttta ctttcttcac
      481 acagtcagct accagcctct tcattgcttt ggggcagttc ttatatagct tactaagatc
      541 tggggaggca tatcctcggc ccaccatgaa gatgatctga tctcggttgt tgatgtgaga
      601 ataaggaagc tcccccgtca tcagttcata caatacgatg ccataggagt agacatccga
      661 ctggaaactg aatgggttgt tatcctgcat tcggatcacc tctggggcca tccagaggac
      721 agagccagta ggttgttcaa cctgctgaga accactccag cgtgacttta ctgttgccaa
      781 accaaaatct ccaattttca ctgttaagcc ttcatggaga aatatattgt tggatttcat
      841 gtctctatgg atgatgttct ttgcatgcaa atagtccatt ccctgagccg tctgccgggc
      901 aatgtcaatt agctggaaca tctgaaactt ggtctcctgg acatgcaggt gtttgtagag
      961 gctgctgccc tcgcaccact gggtcacaat tgccaggttg tcctttgtca tgtaccccat
     1021 gaaaagcaga atgttcacat gccgtgtttt gcgcagaaca gccacctcat tcctgaaggc
     1081 ctggaattgc tctggggttg ggtcgacaac ctttaggatc tttactgcaa catctccgtg
     1141 ccatttaccc ttataaacag ttccaaaaga gcctgaccca atccgagtgg acagcatcac
     1201 ttcactggct tctatttccc aataatagct tgaatctctc tgtccacgag gcctaatttt
     1261 gtttttctcc tgggtcccag atactggtgc ccgctctctt tgtgctggca cgggggtttt
     1321 cggctgtgac cagcctgttg ggctcagatt gttggggcta ctggacaggg ctgaaggtga
     1381 ggctgattcg ctgtgacttc gaattgcatc ctcaatcatc ctgctgtcca caggcagggt
     1441 ggtgctgacc atgtggacat taggtgtgga tgtcgacctc tgcctctggg agagggaacc
     1501 ttcagatgag ggactggagg tgttaaaggt gaaggcgtga ggtgtagaat atctgtgctg
     1561 agaactaaca ggcatcctgg aaacagactc tcgcatacga cgcatagtca aagaaggtag
     1621 tgctgggact ccactatcac caatagtgga atttggaaac aataagagtt gtctgatgtt
     1681 actccagtcc acacacatag taggtacttt ggtgctacag tgctcatgaa atttgtagcc
     1741 acaagtctga catcgaaatc cattgagcag gaatttctga cagatgtcac agaaggcaag
     1801 cttcaggaac gtcttccgag caaagttgtg tgttgtgagg ggaacatgat ccaggaaatc
     1861 tacttgaagt tcttctccaa tcaaagacgc agcatcagta ttccaatcta agcgtgcttt
     1921 tttacctttg tgttcgtgga gaagtctgaa cactgcacag cactctggtt gcaggcccct
     1981 caccttgagt gctttcataa ggcagtcatg caagctcatt ccatttcgca cattgaccac
     2041 tgttctttgc ttgttcggca agaaaacacg gatagtgttg cttgtcttag aaggatctgt
     2101 gagtttgcca tcatctgatg cccggcgctg atagccaaac tgctgaacta ttgtaggaga
     2161 gatgcagctg gagccatcaa acacggcatc tttgaatcca aaaccattgc tgatcgtctt
     2221 ccaagctccc tgtatgtgct ccatggcgat cgcaccgctc gagcctccac ctccgctccc
     2281 gccaccaccg gagctcccac tgttgatggt tactcggaac agcatggagc cgtcgggggt
     2341 gatcaggcgc tcgtcgataa ttttgttgcc gttccacagg gtccctgtta cagtgatctt
     2401 tttgccgtcg aacacggcga tgccttcata cggccgtccg aaatagttca gcatgttcgg
     2461 cgtaaccccg tcgattacca gtgtgccata gggcaggatc accttaaagt gatgatcatc
     2521 cacagggtac accaccttaa acacctcttc gatctgggcc atttggtcgg cgctcagacc
     2581 ttcatacggg atgatgacat ggatgtcgat cttcagggca ttttcaccgc tccggacaat
     2641 cctttggatc ggagttacgg acacggcgag attctgcagc aaactggaca cacctccctg
     2701 ttcaaggact tggtccaggt tgtaggcggc tgtctgttcc cagtccccaa cgaaatcttc
     2761 gagtgtgaag accatggtga gcgctagctg caaaaagaac aagtagcttg tattctatag
     2821 tgtcacctaa atgggcgatc tgacggttca ctaaaccagc tctgcttata taggcctccc
     2881 tggcgcgcca ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     2941 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     3001 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     3061 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     3121 tatcatatgc caagtccgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     3181 tatgcccagt acatgacctt acgggacttt cctacttggc agtacatcta cgtattagtc
     3241 atcgctatta ccatggtgat gcggttttgg cagtacacca atgggcgtgg atagcggttt
     3301 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
     3361 caaaatcaac gggactttcc aaaatgtcgt aataaccccg ccccgttgac gcaaatgggc
     3421 ggtaggcgtg tacggtggga ggtcaaatcg aattctaata ctgttgacaa ttaatcatcg
     3481 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccca ggaggcagat
     3541 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3601 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3661 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3721 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3781 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3841 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3901 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3961 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4021 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4081 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4141 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4201 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4261 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4321 tgacgagttc ttctgattcg aaatgaccga ccaagcgacg cccaaccggt atcagctcac
     4381 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4441 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4501 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4561 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4621 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4681 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4741 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4801 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4861 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4921 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4981 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5041 gtttgcaagc agcagattac gcgcagaaaa aaaggatttc aagaagatcc tttgatcttt
     5101 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5161 ttatcaaaaa ggatcttcac ctagatcctt ttatagtccg gaatcgtcac acaaaaaacc
     5221 aacacacaga tgtaatgaaa ataaagatat tttattgcgg ccatcgtgat ggctagagtc
     5281 taaactcgac ctacctcctt agcgcgcggt cgtgtactgg gccggcggtt gccgctgtcg
     5341 gccagcacct cgacgcagcc gtcggtccat acggctgcgc tccggcgagc gatttgtgtg
     5401 cgaccgacgg tgccggcccc gctgcggacg atggcatcgc agcggccttg agcccaggca
     5461 gcatcgtcga agttgccgtc cacgaggctc tggtagagct gatccaggcc gatgcgcagc
     5521 atgtaggctc gcagacgagg gctgccggcc agctcgggat gccggcgctc gaagtagcga
     5581 gtctgctgct ccatgcaagc cagccagggc cgccagaaga agatgttggc cacctcgtac
     5641 tgactgtccc cgaacatagc ttcggaccag tcgattacgg cggtgatgcg gccgttgtcg
     5701 gtcaggacgt tgttgctgcc gaagtcggca tggacgaggt ggcgcacctc gggacagtct
     5761 tcggcccaca gcatcagttc gtccagggct tgagctacgc tggcggacac ggtgtcgtcc
     5821 atcacggtct gccagtggta gacatgggga tcagcaatgg cgcaaatgaa atcccgccag
     5881 gttgtgtact ggccgatgcc ttggggcccg aatgggccga agccgctggt ttgactgagg
     5941 tcggcggcgg caatagcatc catagcttcg gcgacaggct gtaacacagc aggcagctct
     6001 gtttcgggga ggtcttggag agtaacgcct tgggcgcgtc tactgatgca gtatgtcagg
     6061 ctctcgctga actcgccgat gtccaacact tcggggatgg gtagtgcagc gctggcgaag
     6121 tggcggtaca cgtagcggtc tttgtagaag ccatcagcgc agctattcac ccgcagtaca
     6181 tagccgcgtc cgccgacatc gaagctgaag gctcggctct cttcgccctc cgacaactgc
     6241 atcaggtcgc tcacactgtc gaacttctcg atgagaaatt tttcaacgct ggtagcggtg
     6301 agttcgggct tcttcatggt ggcgctagtg tcagaagaat cgagcttttt gcaaaagcct
     6361 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga ggcggcctcg
     6421 gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc
     6481 ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga
     6541 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct
     6601 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     6661 caccctaact gacacacatt ccacagctgg ttctttccgc ctcagaaggt acctaaccaa
     6721 gttcctcttt cagaggttat ttcaggccat ggtgctgcgc
//
