LOCUS       Exported                5857 bp ds-DNA     circular SYN 15-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5857)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Apr 15, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     pRSG195 designed by Brock F. Binkowski

            

            PCR amplify pF-BiBiTsmN w/ BFBRSG15/BFBRSG14 (PCR #1; 3.9 kbp);

            PCR amplify pF-4B w/ BFBRSG13/BFBRSG16 (PCR #2; 871 bp); 

            2X component Gibson Assembly;

            electroporation of BR610.
FEATURES             Location/Qualifiers
     source          1..5857
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    complement(22..209)
                     /label=SV40\polyA\signal
     misc_feature    complement(300..307)
                     /label=PmeI site
                     /note="Used in Flexi Vector Cloning"
     misc_feature    complement(308..874)
                     /label=KRAS 2B (G12V)
                     /note="KRAS 2B (G12V)"
     misc_feature    complement(839..841)
                     /label=G12V
                     /note="G12V"
     misc_feature    complement(876..883)
                     /label=SgfI Site
                     /note="Used in Flexi Vector Cloning"
     CDS             complement(884..928)
                     /codon_start=1
                     /label=15GS linker
                     /note="15GS linker"
                     /translation="GSSGGGGSGGGGSSG"
     gene            complement(929..1405)
                     /label=LgBiT
                     /note="LgBiT"
     misc_feature    1419..1445
                     /label=B globin leader
                     /note="B globin leader"
     misc_feature    complement(1446..1462)
                     /label=SP6 promoter
                     /note="SP6 promoter"
     misc_feature    complement(1465..1510)
                     /label=Minimal CMV Promoter
     promoter        1520..2110
                     /label=CMV promoter
                     /note="Has EcoICRI site removed. Named changed from ""CMV 
                     Promoter"" to ""CMV immediate-early enhancer/promoter"" 
                     3/21/2011 and J Hartnet's request to fit Sci Com's naming."
     intron          2225..2357
                     /label=chimeric intron
                     /note="Note that this is the actual sequence spliced out. 
                     The 5' donor site includes a 5' GA and the 3' acceptor site
                     adds a 3'G."
     promoter        2401..2420
                     /label=T7 promoter
     CDS             2433..2468
                     /codon_start=1
                     /label=SmBiT
                     /note="SmBiT"
                     /translation="MVTGYRLFEEIL"
     CDS             2469..2513
                     /codon_start=1
                     /label=15GS linker
                     /note="15GS linker"
                     /translation="GSSGGGGSGGGGSSG"
     misc_feature    2514..2521
                     /label=SgfI Site
                     /note="Used in Flexi Vector Cloning"
     misc_feature    2523..2774
                     /label=cRaf RBD
                     /note="cRaf RBD"
     misc_feature    2775..2782
                     /label=PmeI site
                     /note="Used in Flexi Vector Cloning"
     polyA_signal    2911..3132
                     /label=Sv40 late polyadenylation signal
     promoter        3198..3264
                     /label=EM7 bacterial promoter
     CDS             3278..4072
                     /label=KanR
                     /note="Name changed frm ""Neomycin phosphotransferase"" to 
                     ""Neomycin phosphotransferase coding region"" on 3/21/2011 
                     at Jim Hartnet's request to fit Sci Com's naming."
     rep_origin      4227..4263
                     /direction=RIGHT
                     /label=ColE1-derived plasmid replication origin
                     /note="The actual origin is the first base."
     polyA_signal    complement(4945..4992)
                     /label=Synthetic PolyAdenylation Signal
     misc_feature    complement(5016..5414)
                     /label=BlastR
                     /note="BlastR"
     misc_feature    complement(5439..5857)
                     /label=SV40 promoter
                     /note="SV40 promoter"
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 ggtgtttcta gaggatcccc gggtaccgag cccgaattcg
      301 tttaaactta cataattaca cactttgtct ttgacttctt tttcttcttt ttaccatctt
      361 tgctcatctt ttctttatgt tttcgaattt ctcgaactaa tgtatagaag gcatcatcaa
      421 caccctgtct tgtctttgct gatgtttcaa taaaaggaat tccataactt cttgctaagt
      481 cctgagcctg ttttgtgtct actgttctag aaggcaaatc acatttattt cctactagga
      541 ccataggtac atcttcagag tccttaactc ttttaatttg ttctctataa tggtgaatat
      601 cttcaaatga tttagtatta tttatggcaa atacacaaag aaagccctcc ccagtcctca
      661 tgtactggtc cctcattgca ctgtactcct cttgacctgc tgtgtcgaga atatccaaga
      721 gacaggtttc tccatcaatt actacttgct tcctgtagga atcctctatt gttggatcat
      781 attcgtccac aaaatgattc tgaattagct gtatcgtcaa ggcactcttg cctacgccaa
      841 cagctccaac taccacaagt ttatattcag tcatggcgat cgcaccgctc gagcctccac
      901 ctccgctccc gccaccaccg gagctcccac tgttgatggt tactcggaac agcatggagc
      961 cgtcgggggt gatcaggcgc tcgtcgataa ttttgttgcc gttccacagg gtccctgtta
     1021 cagtgatctt tttgccgtcg aacacggcga tgccttcata cggccgtccg aaatagttca
     1081 gcatgttcgg cgtaaccccg tcgattacca gtgtgccata gggcaggatc accttaaagt
     1141 gatgatcatc cacagggtac accaccttaa acacctcttc gatctgggcc atttggtcgg
     1201 cgctcagacc ttcatacggg atgatgacat ggatgtcgat cttcagggca ttttcaccgc
     1261 tccggacaat cctttggatc ggagttacgg acacggcgag attctgcagc aaactggaca
     1321 cacctccctg ttcaaggact tggtccaggt tgtaggcggc tgtctgttcc cagtccccaa
     1381 cgaaatcttc gagtgtgaag accatggtga gcgctagctg caaaaagaac aagtagcttg
     1441 tattctatag tgtcacctaa atgggcgatc tgacggttca ctaaaccagc tctgcttata
     1501 taggcctccc tggcgcgcca ctagttatta atagtaatca attacggggt cattagttca
     1561 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     1621 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     1681 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     1741 acatcaagtg tatcatatgc caagtccgcc ccctattgac gtcaatgacg gtaaatggcc
     1801 cgcctggcat tatgcccagt acatgacctt acgggacttt cctacttggc agtacatcta
     1861 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacacca atgggcgtgg
     1921 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     1981 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aataaccccg ccccgttgac
     2041 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa
     2101 ccgtcagatc actagaagct ttattgcggt agtttatcac agttaaattg ctaacgcagt
     2161 cagtgcttct gacacaacag tctcgaactt aagctgcaga agttggtcgt gaggcactgg
     2221 gcaggtaagt atcaaggtta caagacaggt ttaaggagac caatagaaac tgggcttgtc
     2281 gagacagaga agactcttgc gtttctgata ggcacctatt ggtcttactg acatccactt
     2341 tgcctttctc tccacaggtg tccactccca gttcaattac agctcttaag gctagagtat
     2401 taatacgact cactataggg ctagcgctca ccatggtgac cggctaccgg ctgttcgagg
     2461 agattctcgg gagctccggt ggtggcggga gcggaggtgg aggctcgagc ggtgcgatcg
     2521 ccatgccttc taagacaagc aacactatcc gtgttttctt gccgaacaag caaagaacag
     2581 tggtcaatgt gcgaaatgga atgagcttgc atgactgcct tatgaaagca ctcaaggtga
     2641 ggggcctgca accagagtgc tgtgcagtgt tcagacttct ccacgaacac aaaggtaaaa
     2701 aagcacgctt agattggaat actgatgctg cgtctttgat tggagaagaa cttcaagtag
     2761 atttcctgga tcatgtttaa acgaattcgg gctcggtacc cggggatcct ctagagtcga
     2821 cctgcaggca tgcaagctga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     2881 gctgccaccg ctgagcaagg ccgcttcgag cagacatgat aagatacatt gatgagtttg
     2941 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     3001 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     3061 attttatgtt tcaggttcag ggggagatgt gggaggtttt tttaagcaag taaaacctct
     3121 acaaatgtgg taaaatctta attaagaatt ctaatataac ttcgtatagc atacattata
     3181 cgaagttatg gatccactgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     3241 tacgacaagg tgaggaacta aacccaggag gcagatcatg attgaacaag atggattgca
     3301 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     3361 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     3421 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     3481 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     3541 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     3601 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     3661 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     3721 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     3781 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     3841 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     3901 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     3961 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4021 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gattcgaaat
     4081 gaccgaccaa gcgacgccca accggtatca gctcactcaa aggcggtaat acggttatcc
     4141 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4201 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4261 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4321 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4381 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4441 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4501 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4561 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4621 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     4681 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4741 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4801 agaaaaaaag gatttcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4861 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4921 atccttttat agtccggaat cgtcacacaa aaaaccaaca cacagatgta atgaaaataa
     4981 agatatttta ttgcggccat cgtgatggct agagtttagc cctcccacac ataaccagag
     5041 ggcagcaatt cacgaatccc aactgccgtc ggctgtccat cactgtcctt cactatggct
     5101 ttgatcccag gatgcagatc gagaagcacc tgtcggcacc gtccgcaggg gctcaagatg
     5161 cccctgttct catttccgat cgcgacgata caagtcaggt tgccagctgc cgcagcagca
     5221 gcagtgccca gcaccacgag ttctgcacaa ggtcccccag taaaatgata tacattgaca
     5281 ccagtgaaga tgcggccgtc gctagagaga gctgcgctgg cgacgctgta gtcttcagag
     5341 atggggatgc tgttgattgt agccgttgct ctttcaatga gggtggattc ttcttgagac
     5401 aaaggcttgg ccatggtggc gctagtgtca gaagaatcga gctttttgca aaagcctagg
     5461 cctccaaaaa agcctcctca ctacttctgg aatagctcag aggccgaggc ggcctcggcc
     5521 tctgcataaa taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga
     5581 gttaggggcg ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc
     5641 atgctttgca tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac
     5701 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     5761 cctaactgac acacattcca cagctggttc tttccgcctc agaaggtacc taaccaagtt
     5821 cctctttcag aggttatttc aggccatggt gctgcgc
//
