LOCUS       sequence-472869-        6760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express LgBiT-CRAF(FL) 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  JOURNAL   Unpublished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 Dec 15, 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            "Unpublished"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(FL) BiBiT-Ready Vector
FEATURES             Location/Qualifiers
     source          1..6760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1701..1719)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1702..1719)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        1839..2142
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2297..2317
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        2350..2397
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2429..3223
                     /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(2483..2502)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3093..3112
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(3381..3969)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3461..3480)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4095..4143
                     /note="synthetic polyadenylation signal"
     CDS             complement(4167..5204)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4180..4188
                     /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(5235..5592)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5249..5384
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5401..5592
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5549..5569
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    5664..5745
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5689..5708
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 taggatcttt actgcaacat ctccgtgcca tttaccctta taaacagttc caaaagagcc
       61 tgacccaatc cgagtggaca gcatcacttc actggcttct atttcccaat aatagcttga
      121 atctctctgt ccacgaggcc taattttgtt tttctcctgg gtcccagata ctggtgcccg
      181 ctctctttgt gctggcacgg gggttttcgg ctgtgaccag cctgttgggc tcagattgtt
      241 ggggctactg gacagggctg aaggtgaggc tgattcgctg tgacttcgaa ttgcatcctc
      301 aatcatcctg ctgtccacag gcagggtggt gctgaccatg tggacattag gtgtggatgt
      361 cgacctctgc ctctgggaga gggaaccttc agatgaggga ctggaggtgt taaaggtgaa
      421 ggcgtgaggt gtagaatatc tgtgctgaga actaacaggc atcctggaaa cagactctcg
      481 catacgacgc atagtcaaag aaggtagtgc tgggactcca ctatcaccaa tagtggaatt
      541 tggaaacaat aagagttgtc tgatgttact ccagtccaca cacatagtag gtactttggt
      601 gctacagtgc tcatgaaatt tgtagccaca agtctgacat cgaaatccat tgagcaggaa
      661 tttctgacag atgtcacaga aggcaagctt caggaacgtc ttccgagcaa agttgtgtgt
      721 tgtgagggga acatgatcca ggaaatctac ttgaagttct tctccaatca aagacgcagc
      781 atcagtattc caatctaagc gtgctttttt acctttgtgt tcgtggagaa gtctgaacac
      841 tgcacagcac tctggttgca ggcccctcac cttgagtgct ttcataaggc agtcatgcaa
      901 gctcattcca tttcgcacat tgaccactgt tctttgcttg ttcggcaaga aaacacggat
      961 agtgttgctt gtcttagaag gatctgtgag tttgccatca tctgatgccc ggcgctgata
     1021 gccaaactgc tgaactattg taggagagat gcagctggag ccatcaaaca cggcatcttt
     1081 gaatccaaaa ccattgctga tcgtcttcca agctccctgt atgtgctcca tggcgatcgc
     1141 accgctcgag cctccacctc cgctcccgcc accaccggag ctcccactgt tgatggttac
     1201 tcggaacagc atggagccgt cgggggtgat caggcgctcg tcgataattt tgttgccgtt
     1261 ccacagggtc cctgttacag tgatcttttt gccgtcgaac acggcgatgc cttcatacgg
     1321 ccgtccgaaa tagttcagca tgttcggcgt aaccccgtcg attaccagtg tgccataggg
     1381 caggatcacc ttaaagtgat gatcatccac agggtacacc accttaaaca cctcttcgat
     1441 ctgggccatt tggtcggcgc tcagaccttc atacgggatg atgacatgga tgtcgatctt
     1501 cagggcattt tcaccgctcc ggacaatcct ttggatcgga gttacggaca cggcgagatt
     1561 ctgcagcaaa ctggacacac ctccctgttc aaggacttgg tccaggttgt aggcggctgt
     1621 ctgttcccag tccccaacga aatcttcgag tgtgaagacc atggtgagcg ctagctgcaa
     1681 aaagaacaag tagcttgtat tctatagtgt cacctaaatg ggcgatctga cggttcacta
     1741 aaccagctct gcttatatag gcctccctgg cgcgccacta gttattaata gtaatcaatt
     1801 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     1861 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     1921 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     1981 actgcccact tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc
     2041 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct
     2101 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     2161 tacaccaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     2221 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaat
     2281 aaccccgccc cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt caaatcgaat
     2341 tctaatactg ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag
     2401 gtgaggaact aaacccagga ggcagatcat gattgaacaa gatggattgc acgcaggttc
     2461 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     2521 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     2581 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     2641 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     2701 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     2761 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     2821 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     2881 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     2941 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     3001 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     3061 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     3121 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     3181 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgattcgaaa tgaccgacca
     3241 agcgacgccc aaccggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3301 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     3361 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     3421 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     3481 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3541 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     3601 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3661 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3721 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3781 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     3841 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3901 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3961 ggatttcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4021 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4081 tagtccggaa tcgtcacaca aaaaaccaac acacagatgt aatgaaaata aagatatttt
     4141 attgcggcca tcgtgatggc tagagtctaa actcgaccta cctccttagc gcgcggtcgt
     4201 gtactgggcc ggcggttgcc gctgtcggcc agcacctcga cgcagccgtc ggtccatacg
     4261 gctgcgctcc ggcgagcgat ttgtgtgcga ccgacggtgc cggccccgct gcggacgatg
     4321 gcatcgcagc ggccttgagc ccaggcagca tcgtcgaagt tgccgtccac gaggctctgg
     4381 tagagctgat ccaggccgat gcgcagcatg taggctcgca gacgagggct gccggccagc
     4441 tcgggatgcc ggcgctcgaa gtagcgagtc tgctgctcca tgcaagccag ccagggccgc
     4501 cagaagaaga tgttggccac ctcgtactga ctgtccccga acatagcttc ggaccagtcg
     4561 attacggcgg tgatgcggcc gttgtcggtc aggacgttgt tgctgccgaa gtcggcatgg
     4621 acgaggtggc gcacctcggg acagtcttcg gcccacagca tcagttcgtc cagggcttga
     4681 gctacgctgg cggacacggt gtcgtccatc acggtctgcc agtggtagac atggggatca
     4741 gcaatggcgc aaatgaaatc ccgccaggtt gtgtactggc cgatgccttg gggcccgaat
     4801 gggccgaagc cgctggtttg actgaggtcg gcggcggcaa tagcatccat agcttcggcg
     4861 acaggctgta acacagcagg cagctctgtt tcggggaggt cttggagagt aacgccttgg
     4921 gcgcgtctac tgatgcagta tgtcaggctc tcgctgaact cgccgatgtc caacacttcg
     4981 gggatgggta gtgcagcgct ggcgaagtgg cggtacacgt agcggtcttt gtagaagcca
     5041 tcagcgcagc tattcacccg cagtacatag ccgcgtccgc cgacatcgaa gctgaaggct
     5101 cggctctctt cgccctccga caactgcatc aggtcgctca cactgtcgaa cttctcgatg
     5161 agaaattttt caacgctggt agcggtgagt tcgggcttct tcatggtggc gctagtgtca
     5221 gaagaatcga gctttttgca aaagcctagg cctccaaaaa agcctcctca ctacttctgg
     5281 aatagctcag aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca
     5341 tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc
     5401 gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
     5461 cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg catacttctg
     5521 cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca cagctggttc
     5581 tttccgcctc agaaggtacc taaccaagtt cctctttcag aggttatttc aggccatggt
     5641 gctgcgcaag acgcgtgacg tcggcagtga aaaaaatgct ttatttgtga aatttgtgat
     5701 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     5761 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     5821 ctctacaaat gtggtatggc tgattatgat ccgcggccgc aatacgtcga cgttatcagc
     5881 tgacttcgta cgagagccta ggtgtttcta gaggatcccc gggtaccgag cccgaattcg
     5941 tttaaacgaa gacaggcagc ctcggggacg tggtcagcgt gcaagcattg atatcctcag
     6001 tgtgggctgc ccgatgcaag gatggctcgg aagcgctccg gttgatcttc ggtagagagt
     6061 gttggagcag ctcaatggaa gacaggatct ggggaaaaag aggcctctct tcctttactt
     6121 tcttcacaca gtcagctacc agcctcttca ttgctttggg gcagttctta tatagcttac
     6181 taagatctgg ggaggcatat cctcggccca ccatgaagat gatctgatct cggttgttga
     6241 tgtgagaata aggaagctcc cccgtcatca gttcatacaa tacgatgcca taggagtaga
     6301 catccgactg gaaactgaat gggttgttat cctgcattcg gatcacctct ggggccatcc
     6361 agaggacaga gccagtaggt tgttcaacct gctgagaacc actccagcgt gactttactg
     6421 ttgccaaacc aaaatctcca attttcactg ttaagccttc atggagaaat atattgttgg
     6481 atttcatgtc tctatggatg atgttctttg catgcaaata gtccattccc tgagccgtct
     6541 gccgggcaat gtcaattagc tggaacatct gaaacttggt ctcctggaca tgcaggtgtt
     6601 tgtagaggct gctgccctcg caccactggg tcacaattgc caggttgtcc tttgtcatgt
     6661 accccatgaa aagcagaatg ttcacatgcc gtgttttgcg cagaacagcc acctcattcc
     6721 tgaaggcctg gaattgctct ggggttgggt cgacaacctt
//
