LOCUS       CS1956B08               6617 bp    DNA     circular     31-JAN-2018
pRSG363 (MCS-HiBiT-IRES-luc2), designed by Chris Eggers

Gibson Assembly of
	1. pRSG228 cut with XhoI & PmeI
	2. PCR#1 of luc2 from pGL4.50 with CTERSG3 & 4
	3. GeneBlock GB RSG363

Plate on Kanamycin
Sequence from 1214 to 3630?

One of the C bases in the poly-C tract of the IRES (position 1301)
was changed to T to allow gene block synthesis.
Since T is also a pyrimidine, it may have less effect on the
poly-pyrimidine binding protein thought to bind to the region.
FEATURES             Location/Qualifiers
     promoter        276..866
                     /label=CMV promoter
                     /note="Has EcoICRI site removed. Named changed from
                     %22CMV Promoter%22 to %22CMV immediate-early
                     enhancer/promoter%22 3/21/2011 and J Hartnet's request to
                     fit Sci Com's naming."
     intron          981..1113
                     /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        1157..1176
                     /label=T7 promoter
     polyA_signal    3671..3892
                     /label=Sv40 late polyadenylation signal
     rep_origin      4987..5023
                     /label=ColE1-derived plasmid replication origin
                     /note="The actual origin is the first base."
     polyA_signal    complement(5705..5752)
                     /label=Synthetic PolyAdenylation Signal Sequence (from
                     pCiNeo vectors)
     misc_feature    complement(22..209)
                     /label=SV40\polyA\signal
     misc_feature    complement(3916..3949)
                     /note="Cre site"
     promoter        3958..4024
                     /label=EM7 bacterial promoter
     CDS             4038..4832
                     /label=KanR
                     /note="Name changed frm %22Neomycin phosphotransferase%22
                     to %22Neomycin phosphotransferase coding region%22 on
                     3/21/2011 at Jim Hartnet's request to fit Sci Com's
                     naming."
     misc_feature    complement(5776..6174)
                     /note="BlastR"
     misc_feature    complement(6199..6617)
                     /note="SV40 promoter"
     misc_feature    complement(1177..1184)
                     /note="SgfI"
     gene            1262..1294
                     /note="HiBiT"
                     /translation="MVSGWRLFKKIS"
     misc_feature    1238..1261
                     /note="8 GS linker"
     misc_feature    1939..3588
                     /note="luc2"
     misc_feature    1329..1938
                     /note="EMCV IRES"
     misc_feature    1942..1968
                     /note="CTERSG3"
     misc_feature    complement(3573..3617)
                     /note="CTERSG4"
     misc_feature    1379..1379
                     /note="C > T mutation to allow synthesis"
     source          1..6617
                     /dnas_title="CS1956B08 pBiT4.1-C HiBiT-IRES-luc2"
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 GGATTATGGC GCGCCACTAG TTATTAATAG TAATCAATTA
      301 CGGGGTCATT AGTTCATAGC CCATATATGG AGTTCCGCGT TACATAACTT ACGGTAAATG
      361 GCCCGCCTGG CTGACCGCCC AACGACCCCC GCCCATTGAC GTCAATAATG ACGTATGTTC
      421 CCATAGTAAC GCCAATAGGG ACTTTCCATT GACGTCAATG GGTGGAGTAT TTACGGTAAA
      481 CTGCCCACTT GGCAGTACAT CAAGTGTATC ATATGCCAAG TCCGCCCCCT ATTGACGTCA
      541 ATGACGGTAA ATGGCCCGCC TGGCATTATG CCCAGTACAT GACCTTACGG GACTTTCCTA
      601 CTTGGCAGTA CATCTACGTA TTAGTCATCG CTATTACCAT GGTGATGCGG TTTTGGCAGT
      661 ACACCAATGG GCGTGGATAG CGGTTTGACT CACGGGGATT TCCAAGTCTC CACCCCATTG
      721 ACGTCAATGG GAGTTTGTTT TGGCACCAAA ATCAACGGGA CTTTCCAAAA TGTCGTAATA
      781 ACCCCGCCCC GTTGACGCAA ATGGGCGGTA GGCGTGTACG GTGGGAGGTC TATATAAGCA
      841 GAGCTGGTTT AGTGAACCGT CAGATCACTA GAAtCTTTAT TGCGGTAGTT TATCACAGTT
      901 AAATTGCTAA CGCAGTCAGT GCTTCTGACA CAACAGTCTC GAACTTAAGC TGCAGAAGTT
      961 GGTCGTGAGG CACTGGGCAG GTAAGTATCA AGGTTACAAG ACAGGTTTAA GGAGACCAAT
     1021 AGAAACTGGG CTTGTCGAGA CAGAGAAGAC TCTTGCGTTT CTGATAGGCA CCTATTGGTC
     1081 TTACTGACAT CCACTTTGCC TTTCTCTCCA CAGGTGTCCA CTCCCAGTTC AATTACAGCT
     1141 CTTAAGGCTA GAGTATTAAT ACGACTCACT ATAGGGGCGA TCGCaacAAG CTTcgggaAG
     1201 ATCTggaGCT AGCggcgggG GATCCggtgg GAATTCtggC TCGAGcggtg ggagctccgg
     1261 tGTGAGCGGC TGGCGGCTGT TCAAGAAGAT TAGCtgaccg accaagcgac gcccaacctg
     1321 ccatcacgga ccacaacggt ttccctctag cgggatcaat tccgcccctc tccctccctc
     1381 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
     1441 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
     1501 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg
     1561 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     1621 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     1681 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     1741 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     1801 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt
     1861 gtttagtcga ggttaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt
     1921 GAAAAACACG ATGATAATAT GGAAGATGCC AAAAACATTA AGAAGGGCCC AGCGCCATTC
     1981 TACCCACTCG AAGACGGGAC CGCCGGCGAG CAGCTGCACA AAGCCATGAA GCGCTACGCC
     2041 CTGGTGCCCG GCACCATCGC CTTTACCGAC GCACATATCG AGGTGGACAT TACCTACGCC
     2101 GAGTACTTCG AGATGAGCGT TCGGCTGGCA GAAGCTATGA AGCGCTATGG GCTGAATACA
     2161 AACCATCGGA TCGTGGTGTG CAGCGAGAAT AGCTTGCAGT TCTTCATGCC CGTGTTGGGT
     2221 GCCCTGTTCA TCGGTGTGGC TGTGGCCCCA GCTAACGACA TCTACAACGA GCGCGAGCTG
     2281 CTGAACAGCA TGGGCATCAG CCAGCCCACC GTCGTATTCG TGAGCAAGAA AGGGCTGCAA
     2341 AAGATCCTCA ACGTGCAAAA GAAGCTACCG ATCATACAAA AGATCATCAT CATGGATAGC
     2401 AAGACCGACT ACCAGGGCTT CCAAAGCATG TACACCTTCG TGACTTCCCA TTTGCCACCC
     2461 GGCTTCAACG AGTACGACTT CGTGCCCGAG AGCTTCGACC GGGACAAAAC CATCGCCCTG
     2521 ATCATGAACA GTAGTGGCAG TACCGGATTG CCCAAGGGCG TAGCCCTACC GCACCGCACC
     2581 GCTTGTGTCC GATTCAGTCA TGCCCGCGAC CCCATCTTCG GCAACCAGAT CATCCCCGAC
     2641 ACCGCTATCC TCAGCGTGGT GCCATTTCAC CACGGCTTCG GCATGTTCAC CACGCTGGGC
     2701 TACTTGATCT GCGGCTTTCG GGTCGTGCTC ATGTACCGCT TCGAGGAGGA GCTATTCTTG
     2761 CGCAGCTTGC AAGACTATAA GATTCAATCT GCCCTGCTGG TGCCCACACT ATTTAGCTTC
     2821 TTCGCTAAGA GCACTCTCAT CGACAAGTAC GACCTAAGCA ACTTGCACGA GATCGCCAGC
     2881 GGCGGGGCGC CGCTCAGCAA GGAGGTAGGT GAGGCCGTGG CCAAACGCTT CCACCTACCA
     2941 GGCATCCGCC AGGGCTACGG CCTGACAGAA ACAACCAGCG CCATTCTGAT CACCCCCGAA
     3001 GGGGACGACA AGCCTGGCGC AGTAGGCAAG GTGGTGCCCT TCTTCGAGGC TAAGGTGGTG
     3061 GACTTGGACA CCGGTAAGAC ACTGGGTGTG AACCAGCGCG GCGAGCTGTG CGTCCGTGGC
     3121 CCCATGATCA TGAGCGGCTA CGTTAACAAC CCCGAGGCTA CAAACGCTCT CATCGACAAG
     3181 GACGGCTGGC TGCACAGCGG CGACATCGCC TACTGGGACG AGGACGAGCA CTTCTTCATC
     3241 GTGGACCGGC TGAAGAGCCT GATCAAATAC AAGGGCTACC AGGTAGCCCC AGCCGAACTG
     3301 GAGAGCATCC TGCTGCAACA CCCCAACATC TTCGACGCCG GGGTCGCCGG CCTGCCCGAC
     3361 GACGATGCCG GCGAGCTGCC CGCCGCAGTC GTCGTGCTGG AACACGGTAA AACCATGACC
     3421 GAGAAGGAGA TCGTGGACTA TGTGGCCAGC CAGGTTACAA CCGCCAAGAA GCTGCGCGGT
     3481 GGTGTTGTGT TCGTGGACGA GGTGCCTAAA GGACTGACCG GCAAGTTGGA CGCCCGCAAG
     3541 ATCCGCGAGA TTCTCATTAA GGCCAAGAAG GGCGGCAAGA TCGCCGTGTA AgtttaaacG
     3601 GCCGCGACTC TAGACTGCAG GCATGCAAGC TGATTTGGCT GCTGCCACCG CTGAGCAAGG
     3661 CCGCTTCGAG CAGACATGAT AAGATACATT GATGAGTTTG GACAAACCAC AACTAGAATG
     3721 CAGTGAAAAA AATGCTTTAT TTGTGAAATT TGTGATGCTA TTGCTTTATT TGTAACCATT
     3781 ATAAGCTGCA ATAAACAAGT TAACAACAAC AATTGCATTC ATTTTATGTT TCAGGTTCAG
     3841 GGGGAGATGT GGGAGGTTTT TTTAAGCAAG TAAAACCTCT ACAAATGTGG TAAAATCTTA
     3901 ATTAAGAcTT CTAATATAAC TTCGTATAGC ATACATTATA CGAAGTTATG GtTCCACTGT
     3961 TGACAATTAA TCATCGGCAT AGTATATCGG CATAGTATAA TACGACAAGG TGAGGAACTA
     4021 AACCCAGGAG GCAGATCATG ATTGAACAAG ATGGATTGCA CGCAGGTTCT CCGGCCGCTT
     4081 GGGTGGAGAG GCTATTCGGC TATGACTGGG CACAACAGAC AATCGGCTGC TCTGATGCCG
     4141 CCGTGTTCCG GCTGTCAGCG CAGGGGCGCC CGGTTCTTTT TGTCAAGACC GACCTGTCCG
     4201 GTGCCCTGAA TGAACTGCAG GACGAGGCAG CGCGGCTATC GTGGCTGGCC ACGACGGGCG
     4261 TTCCTTGCGC AGCTGTGCTC GACGTTGTCA CTGAAGCGGG AAGGGACTGG CTGCTATTGG
     4321 GCGAAGTGCC GGGGCAGGAT CTCCTGTCAT CTCACCTTGC TCCTGCCGAG AAAGTATCCA
     4381 TCATGGCTGA TGCAATGCGG CGGCTGCATA CGCTTGATCC GGCTACCTGC CCATTCGACC
     4441 ACCAAGCGAA ACATCGCATC GAGCGAGCAC GTACTCGGAT GGAAGCCGGT CTTGTCGATC
     4501 AGGATGATCT GGACGAAGAG CATCAGGGGC TCGCGCCAGC CGAACTGTTC GCCAGGCTCA
     4561 AGGCGCGCAT GCCCGACGGC GAGGATCTCG TCGTGACCCA TGGCGATGCC TGCTTGCCGA
     4621 ATATCATGGT GGAAAATGGC CGCTTTTCTG GATTCATCGA CTGTGGCCGG CTGGGTGTGG
     4681 CGGACCGCTA TCAGGACATA GCGTTGGCTA CCCGTGATAT TGCTGAAGAG CTTGGCGGCG
     4741 AATGGGCTGA CCGCTTCCTC GTGCTTTACG GTATCGCCGC TCCCGATTCG CAGCGCATCG
     4801 CCTTCTATCG CCTTCTTGAC GAGTTCTTCT GATTCGAAAT GACCGACCAA GCGACGCCCA
     4861 ACCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC
     4921 AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT
     4981 GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG
     5041 TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC
     5101 CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC
     5161 TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT
     5221 CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT
     5281 ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC
     5341 AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA
     5401 GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG CTCTGCTGAA
     5461 GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG
     5521 TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATTTCAAGA
     5581 AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG
     5641 GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAT AGTCCGGAAT
     5701 CGTCACACAA AAAACCAACA CACAGATGTA ATGAAAATAA AGATATTTTA TTGCGGCCAT
     5761 CGTGATGGCT AGAGTTTAGC CCTCCCACAC ATAACCAGAG GGCAGCAATT CACGAATCCC
     5821 AACTGCCGTC GGCTGTCCAT CACTGTCCTT CACTATGGCT TTGATCCCAG GATGCAGATC
     5881 GAGAAGCACC TGTCGGCACC GTCCGCAGGG GCTCAAGATG CCCCTGTTCT CATTTCCGAT
     5941 CGCGACGATA CAAGTCAGGT TGCCAGCTGC CGCAGCAGCA GCAGTGCCCA GCACCACGAG
     6001 TTCTGCACAA GGTCCCCCAG TAAAATGATA TACATTGACA CCAGTGAAGA TGCGGCCGTC
     6061 GCTAGAGAGA GCTGCGCTGG CGACGCTGTA GTCTTCAGAG ATGGGGATGC TGTTGATTGT
     6121 AGCCGTTGCT CTTTCAATGA GGGTGGATTC TTCTTGAGAC AAAGGCTTGG CCATggtggc
     6181 gctagtgtca gaagaatcGA GCTTTTTGCA AAAGCCTAGG CCTCCAAAAA AGCCTCCTCA
     6241 CTACTTCTGG AATAGCTCAG AGGCCGAGGC GGCCTCGGCC TCTGCATAAA TAAAAAAAAT
     6301 TAGTCAGCCA TGGGGCGGAG AATGGGCGGA ACTGGGCGGA GTTAGGGGCG GGATGGGCGG
     6361 AGTTAGGGGC GGGACTATGG TTGCTGACTA ATTGAGATGC ATGCTTTGCA TACTTCTGCC
     6421 TGCTGGGGAG CCTGGGGACT TTCCACACCT GGTTGCTGAC TAATTGAGAT GCATGCTTTG
     6481 CATACTTCTG CCTGCTGGGG AGCCTGGGGA CTTTCCACAC CCTAACTGAC ACACATTCCA
     6541 CAGCTGGTTC TTTCCGCCTC AGAAGGTACC TAACCAAGTT CCTCTTTCAG AGGTTATTTC
     6601 AGGCCATGGT GCTGCGC
//

