LOCUS       sequence-476610-        5492 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  Express LgBiT-NRAS (Q61R) 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 5492)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 5492)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: LgBiT-NRAS (Q61R) BiBiT-Ready Vector
FEATURES             Location/Qualifiers
     source          1..5492
                     /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"
     primer_bind     275..293
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     promoter        complement(1546..1564)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1547..1564)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        1684..1987
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2142..2162
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        2195..2242
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2274..3068
                     /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(2328..2347)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2938..2957
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(3226..3814)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3306..3325)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3940..3988
                     /note="synthetic polyadenylation signal"
     CDS             complement(4012..5049)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4025..4033
                     /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(5080..5437)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5094..5229
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5246..5437
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5394..5414
                     /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 ccccaagggg ttatgctagt tattgctcag cggtggcagc
      301 agccaactca gcttcctttc gggctttgtt agcagccgga tcagcttgca tgcctgcagg
      361 tcgactctag aggatccccg ggtaccgagc ccgaattcgt ttaaacttac atcaccacac
      421 atggcaatcc catacaaccc tgagtcccat catcactgct gttgagtttt ttcattcggt
      481 actggcgtat ttctcttacc agtgtgtaaa aagcatcttc aacaccctgt ctggtcttgg
      541 ctgaggtttc aatgaatgga atcccgtaac tcttggccag ttcgtgggct tgttttgtat
      601 caactgtcct tgttggcaaa tcacacttgt ttcccactag caccataggt acatcatccg
      661 agtcttttac tcgcttaatc tgctccctgt agaggttaat atccgcaaat gacttgctat
      721 tattgatggc aaatacacag aggaagcctt cgcctgtcct catgtattgg tctctcatgg
      781 cactgtactc ttctcttcca gctgtatcca gtatgtccaa caaacaggtt tcaccatcta
      841 taaccacttg ttttctgtaa gaatcctcta tggtgggatc atattcatct acaaagtggt
      901 tctggattag ctggattgtc agtgcgcttt tcccaacacc acctgctcca accaccacca
      961 gtttgtactc agtcatggcg atcgcaccgc tcgagcctcc acctccgctc ccgccaccac
     1021 cggagctccc actgttgatg gttactcgga acagcatgga gccgtcgggg gtgatcaggc
     1081 gctcgtcgat aattttgttg ccgttccaca gggtccctgt tacagtgatc tttttgccgt
     1141 cgaacacggc gatgccttca tacggccgtc cgaaatagtt cagcatgttc ggcgtaaccc
     1201 cgtcgattac cagtgtgcca tagggcagga tcaccttaaa gtgatgatca tccacagggt
     1261 acaccacctt aaacacctct tcgatctggg ccatttggtc ggcgctcaga ccttcatacg
     1321 ggatgatgac atggatgtcg atcttcaggg cattttcacc gctccggaca atcctttgga
     1381 tcggagttac ggacacggcg agattctgca gcaaactgga cacacctccc tgttcaagga
     1441 cttggtccag gttgtaggcg gctgtctgtt cccagtcccc aacgaaatct tcgagtgtga
     1501 agaccatggt gagcgctagc tgcaaaaaga acaagtagct tgtattctat agtgtcacct
     1561 aaatgggcga tctgacggtt cactaaacca gctctgctta tataggcctc cctggcgcgc
     1621 cactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     1681 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     1741 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     1801 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     1861 gccaagtccg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     1921 gtacatgacc ttacgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     1981 taccatggtg atgcggtttt ggcagtacac caatgggcgt ggatagcggt ttgactcacg
     2041 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     2101 acgggacttt ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg gcggtaggcg
     2161 tgtacggtgg gaggtcaaat cgaattctaa tactgttgac aattaatcat cggcatagta
     2221 tatcggcata gtataatacg acaaggtgag gaactaaacc caggaggcag atcatgattg
     2281 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     2341 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     2401 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     2461 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     2521 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     2581 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     2641 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     2701 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     2761 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     2821 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     2881 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     2941 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     3001 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     3061 tcttctgatt cgaaatgacc gaccaagcga cgcccaaccg gtatcagctc actcaaaggc
     3121 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3181 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3241 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3301 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3361 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3421 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3481 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3541 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3601 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3661 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3721 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3781 gcagcagatt acgcgcagaa aaaaaggatt tcaagaagat cctttgatct tttctacggg
     3841 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3901 aaggatcttc acctagatcc ttttatagtc cggaatcgtc acacaaaaaa ccaacacaca
     3961 gatgtaatga aaataaagat attttattgc ggccatcgtg atggctagag tctaaactcg
     4021 acctacctcc ttagcgcgcg gtcgtgtact gggccggcgg ttgccgctgt cggccagcac
     4081 ctcgacgcag ccgtcggtcc atacggctgc gctccggcga gcgatttgtg tgcgaccgac
     4141 ggtgccggcc ccgctgcgga cgatggcatc gcagcggcct tgagcccagg cagcatcgtc
     4201 gaagttgccg tccacgaggc tctggtagag ctgatccagg ccgatgcgca gcatgtaggc
     4261 tcgcagacga gggctgccgg ccagctcggg atgccggcgc tcgaagtagc gagtctgctg
     4321 ctccatgcaa gccagccagg gccgccagaa gaagatgttg gccacctcgt actgactgtc
     4381 cccgaacata gcttcggacc agtcgattac ggcggtgatg cggccgttgt cggtcaggac
     4441 gttgttgctg ccgaagtcgg catggacgag gtggcgcacc tcgggacagt cttcggccca
     4501 cagcatcagt tcgtccaggg cttgagctac gctggcggac acggtgtcgt ccatcacggt
     4561 ctgccagtgg tagacatggg gatcagcaat ggcgcaaatg aaatcccgcc aggttgtgta
     4621 ctggccgatg ccttggggcc cgaatgggcc gaagccgctg gtttgactga ggtcggcggc
     4681 ggcaatagca tccatagctt cggcgacagg ctgtaacaca gcaggcagct ctgtttcggg
     4741 gaggtcttgg agagtaacgc cttgggcgcg tctactgatg cagtatgtca ggctctcgct
     4801 gaactcgccg atgtccaaca cttcggggat gggtagtgca gcgctggcga agtggcggta
     4861 cacgtagcgg tctttgtaga agccatcagc gcagctattc acccgcagta catagccgcg
     4921 tccgccgaca tcgaagctga aggctcggct ctcttcgccc tccgacaact gcatcaggtc
     4981 gctcacactg tcgaacttct cgatgagaaa tttttcaacg ctggtagcgg tgagttcggg
     5041 cttcttcatg gtggcgctag tgtcagaaga atcgagcttt ttgcaaaagc ctaggcctcc
     5101 aaaaaagcct cctcactact tctggaatag ctcagaggcc gaggcggcct cggcctctgc
     5161 ataaataaaa aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag
     5221 gggcgggatg ggcggagtta ggggcgggac tatggttgct gactaattga gatgcatgct
     5281 ttgcatactt ctgcctgctg gggagcctgg ggactttcca cacctggttg ctgactaatt
     5341 gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc cacaccctaa
     5401 ctgacacaca ttccacagct ggttctttcc gcctcagaag gtacctaacc aagttcctct
     5461 ttcagaggtt atttcaggcc atggtgctgc gc
//
