LOCUS Exported 5862 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Backbone for transient transfection of Massively Parallel Reporter Assays (MPRA) libraries, pPGK-H2Kk for physical selection of transfected cells. ACCESSION . VERSION . KEYWORDS pMPRA2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5862) AUTHORS Melnikov A, Zhang X, Rogov P, Wang L, Mikkelsen TS TITLE Massively parallel reporter assays in cultured Mammalian cells. JOURNAL J Vis Exp. 2014 Aug 17;(90). doi: 10.3791/51719. PUBMED 25177895 REFERENCE 2 (bases 1 to 5862) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5862 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 40..164 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 189..219 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 273..932 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(339..358) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 1274..1579 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 1458..1477 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(1620..1744) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" polyA_signal 1798..1919 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1809..1828) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1863..1882 /label=SV40pA-R /note="SV40 polyA, reverse primer" promoter 2021..2521 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 2440..2458 /label=hPGK-F /note="Human PGK promoter, forward primer" regulatory 2522..2531 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" polyA_signal 3574..3622 /note="synthetic polyadenylation signal" primer_bind complement(3789..3806) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3960..4548) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4040..4059) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4749..5609) /codon_start=1 /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" polyA_signal 5709..5757 /note="synthetic polyadenylation signal" misc_feature 5771..5862 /label=pause site /note="RNA polymerase II transcriptional pause signal from the human alpha-2 globin gene" primer_bind 5811..5830 /label=RVprimer3 /note="pGL3 vector, forward primer" ORIGIN 1 ggcctaactg gccagtgtgg tggaattctg cagatatcaa caagtttgta caaaaaagct 61 gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa 121 cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc gcattaggca 181 ccccaggctt tacactttat gcttccggct cgtataatgt gtggattttg agttaggatc 241 cgtcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca 301 ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc 361 aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga 421 aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc 481 atccggaatt ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc 541 cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc 601 acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa 661 acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct 721 gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg 781 ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc 841 aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac 901 agtactgcga tgagtggcag ggcggggcgt aaagatctgg atccggctta ctaaaagcca 961 gataacagta tgcgtatttg cgcgctgatt tttgcggtat aagaatatat actgatatgt 1021 atacccgaag tatgtcaaaa agaggtatgc tatgaagcag cgtattacag tgacagttga 1081 cagcgacagc tatcagttgc tcaaggcata tatgatgtca atatctccgg tctggtaagc 1141 acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag 1201 gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg gctcttttgc tgacgagaac 1261 aggggctggt gaaatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct 1321 gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct 1381 ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat 1441 cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat 1501 cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct 1561 gatgttctgg ggaatataaa tgtcaggctc ccttatacac agccagtctg caggtcgacc 1621 atagtgactg gatatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct 1681 aatttaatat attgatattt atatcatttt acgtttctcg ttcagctttc ttgtacaaag 1741 tggttggcct cggcggccaa gctagtcggg gcggccggcc gcttcgagca gacatgataa 1801 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt 1861 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta 1921 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt 1981 aaagcaagta aaacctctac aaatgtggta aaatcgataa ttgcgccttt tccaaggcag 2041 ccctgggttt gcgcagggac gcggctgctc tgggcgtggt tccgggaaac gcagcggcgc 2101 cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc gtcacccgga tcttcgccgc 2161 tacccttgtg ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg aaggttcctt 2221 gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta gtaccctcgc 2281 agacggacag cgccagggag caatggcagc gcgccgaccg cgatgggctg tggccaatag 2341 cggctgctca gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc gggaggcggg 2401 gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat tctgcaagcc 2461 tccggagcgc acgtcggcag tcggctccct cgttgaccga atcaccgacc tctctcccca 2521 ggccaccatg gcaccctgca tgctgctcct gctgttggcg gctgccctgg ccccgactca 2581 gacccgcgcg ggcccacatt cgctgaggta tttccacacc gccgtgtccc ggcccggcct 2641 cgggaagccc cggttcatct ctgtcggcta cgtggacgac acgcagttcg tgcgcttcga 2701 cagcgacgcg gagaatccga ggtatgagcc gcgggtgcgg tggatggagc aggtggagcc 2761 cgagtattgg gagcggaaca cgcagatcgc caagggcaat gagcagattt tccgagtgaa 2821 cctgaggacc gcgctgcgct actacaacca gagcgcgggc ggctctcaca cgttccaacg 2881 gatgtacggc tgtgaggtgg ggtcggactg gcgcctcctc cgcgggtacg agcagtacgc 2941 atacgacggc tgcgattaca tcgccctgaa cgaagacctg aaaacgtgga cggcggccga 3001 catggcggcg ctgatcacca aacacaagtg ggagcaggct ggtgatgcag agagagaccg 3061 ggcctacctg gagggcacgt gcgtggagtg gctccgcaga tacctgcagc tcgggaacgc 3121 gacgctgccg cgcacagatt ccccaaaggc ccatgtgacc cgtcacagca gacctgaaga 3181 taaagtcacc ctgaggtgct gggccctggg cttctaccct gctgacatca ccctgacctg 3241 gcagttgaat ggggaggagc tgacccagga catggagctt gtggagacca ggcctgcagg 3301 ggatggaacc ttccagaagt gggcatctgt ggtggtgcct cttgggaagg agcagtatta 3361 cacatgccat gtgtaccatc aggggctgcc tgagcccctc accctgagat gggagcctcc 3421 tccatccact gtctccaaca cggtaatcat tgctgttctg gttgtccttg gagctgcaat 3481 agtcactgga gctgtggtgg cttttgtgat gaagatgaga aggagaaaca caggtggaaa 3541 aggagggtga actcttgaac cggtcatggc cgcaataaaa tatctttatt ttcattacat 3601 ctgtgtgttg gttttttgtg tgggatccgt cgaccgatgc ccttgagagc cttcaaccca 3661 gtcagctcct tttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg 3721 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct 3781 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 3841 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 3901 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 3961 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 4021 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 4081 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 4141 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 4201 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 4261 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 4321 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 4381 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 4441 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 4501 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 4561 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 4621 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 4681 tcaatctaaa gtatatatga gtaaacttgg tctgacagcg gccgcaaatg ctaaaccact 4741 gcagtggttt accagtgctt gatcagtgag gcaccgatct cagcgatctg cctatttcgt 4801 tcgtccatag tggcctgact ccccgtcgtg tagatcacta cgattcgtga gggcttacca 4861 tcaggcccca gcgcagcaat gatgccgcga gagccgcgtt caccggcccc cgatttgtca 4921 gcaatgaacc agccagcagg gagggccgag cgaagaagtg gtcctgctac tttgtccgcc 4981 tccatccagt ctatgagctg ctgtcgtgat gctagagtaa gaagttcgcc agtgagtagt 5041 ttccgaagag ttgtggccat tgctactggc atcgtggtat cacgctcgtc gttcggtatg 5101 gcttcgttca actctggttc ccagcggtca agccgggtca catgatcacc catattatga 5161 agaaatgcag tcagctcctt agggcctccg atcgttgtca gaagtaagtt ggccgcggtg 5221 ttgtcgctca tggtaatggc agcactacac aattctctta ccgtcatgcc atccgtaaga 5281 tgcttttccg tgaccggcga gtactcaacc aagtcgtttt gtgagtagtg tatacggcga 5341 ccaagctgct cttgcccggc gtctatacgg gacaacaccg cgccacatag cagtactttg 5401 aaagtgctca tcatcgggaa tcgttcttcg gggcggaaag actcaaggat cttgccgcta 5461 ttgagatcca gttcgatata gcccactctt gcacccagtt gatcttcagc atcttttact 5521 ttcaccagcg tttcggggtg tgcaaaaaca ggcaagcaaa atgccgcaaa gaagggaatg 5581 agtgcgacac gaaaatgttg gatgctcatt cctttttcaa tattattgaa gcatttatca 5641 gggttactag tacgtctctc aaggataagt aagtaatatt aaggtacggg aggtattgga 5701 caggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgaat 5761 cgatagtact aacatacgct ctccatcaaa acaaaacgaa acaaaacaaa ctagcaaaat 5821 aggctgtccc cagtgcaagt gcaggtgcca gaacatttct ct //