LOCUS       sequence-451373-        4244 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Basic backbone for transient transfection of Massively Parallel 
            Reporter Assay (MPRA) libraries.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4244)
  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 4244)
  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: "J Vis 
            Exp."; date: "2014-08-17"; volume: "(90). doi"; pages: " 
            10.3791/51719"
COMMENT     Sequence Label: pMPRA1
FEATURES             Location/Qualifiers
     source          1..4244
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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        join(189..194,195..212,213..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"
     primer_bind     complement(2167..2184)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2338..2926)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2418..2437)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3126..3917,3918..3986))
                     /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    4091..4139
                     /note="synthetic polyadenylation signal"
     misc_feature    4153..4244
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4193..4212
                     /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 ggatccgtcg accgatgccc
     2041 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc
     2101 gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc
     2161 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     2221 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2281 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2341 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2401 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2461 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2521 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2581 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     2641 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     2701 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     2761 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     2821 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     2881 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2941 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3001 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3061 aatctaaagt atatatgagt aaacttggtc tgacagcggc cgcaaatgct aaaccactgc
     3121 agtggttacc agtgcttgat cagtgaggca ccgatctcag cgatctgcct atttcgttcg
     3181 tccatagtgg cctgactccc cgtcgtgtag atcactacga ttcgtgaggg cttaccatca
     3241 ggccccagcg cagcaatgat gccgcgagag ccgcgttcac cggcccccga tttgtcagca
     3301 atgaaccagc cagcagggag ggccgagcga agaagtggtc ctgctacttt gtccgcctcc
     3361 atccagtcta tgagctgctg tcgtgatgct agagtaagaa gttcgccagt gagtagtttc
     3421 cgaagagttg tggccattgc tactggcatc gtggtatcac gctcgtcgtt cggtatggct
     3481 tcgttcaact ctggttccca gcggtcaagc cgggtcacat gatcacccat attatgaaga
     3541 aatgcagtca gctccttagg gcctccgatc gttgtcagaa gtaagttggc cgcggtgttg
     3601 tcgctcatgg taatggcagc actacacaat tctcttaccg tcatgccatc cgtaagatgc
     3661 ttttccgtga ccggcgagta ctcaaccaag tcgttttgtg agtagtgtat acggcgacca
     3721 agctgctctt gcccggcgtc tatacgggac aacaccgcgc cacatagcag tactttgaaa
     3781 gtgctcatca tcgggaatcg ttcttcgggg cggaaagact caaggatctt gccgctattg
     3841 agatccagtt cgatatagcc cactcttgca cccagttgat cttcagcatc ttttactttc
     3901 accagcgttt cggggtgtgc aaaaacaggc aagcaaaatg ccgcaaagaa gggaatgagt
     3961 gcgacacgaa aatgttggat gctcatactc gtcctttttc aatattattg aagcatttat
     4021 cagggttact agtacgtctc tcaaggataa gtaagtaata ttaaggtacg ggaggtattg
     4081 gacaggccgc aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga
     4141 atcgatagta ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa
     4201 ataggctgtc cccagtgcaa gtgcaggtgc cagaacattt ctct
//
