LOCUS       sequence-451374-        6623 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for transient transfection of Massively Parallel Reporter 
            Assays (MPRA) libraries, pPGK-H2Kk for physical selection of 
            transfected cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6623)
  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 6623)
  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: pMPRA2
FEATURES             Location/Qualifiers
     source          1..6623
                     /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"
     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
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3574..3622
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(3769..3786)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3940..4528)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4020..4039)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4728..5519,5520..5588))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     mobile_element  5738..6505
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     misc_feature    6532..6623
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     6572..6591
                     /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 tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc
     3721 tttatcatgc aactcgtagg acaggtgccg gcagcgctct tccgcttcct cgctcactga
     3781 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3841 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3901 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3961 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4021 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4081 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4141 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     4201 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4261 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4321 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4381 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4441 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4501 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4561 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     4621 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     4681 gtaaacttgg tctgacagcg gccgcaaatg ctaaaccact gcagtggtta ccagtgcttg
     4741 atcagtgagg caccgatctc agcgatctgc ctatttcgtt cgtccatagt ggcctgactc
     4801 cccgtcgtgt agatcactac gattcgtgag ggcttaccat caggccccag cgcagcaatg
     4861 atgccgcgag agccgcgttc accggccccc gatttgtcag caatgaacca gccagcaggg
     4921 agggccgagc gaagaagtgg tcctgctact ttgtccgcct ccatccagtc tatgagctgc
     4981 tgtcgtgatg ctagagtaag aagttcgcca gtgagtagtt tccgaagagt tgtggccatt
     5041 gctactggca tcgtggtatc acgctcgtcg ttcggtatgg cttcgttcaa ctctggttcc
     5101 cagcggtcaa gccgggtcac atgatcaccc atattatgaa gaaatgcagt cagctcctta
     5161 gggcctccga tcgttgtcag aagtaagttg gccgcggtgt tgtcgctcat ggtaatggca
     5221 gcactacaca attctcttac cgtcatgcca tccgtaagat gcttttccgt gaccggcgag
     5281 tactcaacca agtcgttttg tgagtagtgt atacggcgac caagctgctc ttgcccggcg
     5341 tctatacggg acaacaccgc gccacatagc agtactttga aagtgctcat catcgggaat
     5401 cgttcttcgg ggcggaaaga ctcaaggatc ttgccgctat tgagatccag ttcgatatag
     5461 cccactcttg cacccagttg atcttcagca tcttttactt tcaccagcgt ttcggggtgt
     5521 gcaaaaacag gcaagcaaaa tgccgcaaag aagggaatga gtgcgacacg aaaatgttgg
     5581 atgctcatac tcgtcctttt tcaatattat tgaagcattt atcagggtta ctagtacgtc
     5641 tctcaaggat aagtaagtaa tattaaggta cgggaggtat tggacaggcc gcaataaaat
     5701 atctttattt tcattacatc tgtgtgttgg ttttttgggt aatgactcca acttattgat
     5761 agtgttttat gttcagataa tgcccgatga ctttgtcatg cagctccacc gattttgaga
     5821 acgacagcga cttccgtccc agccgtgcca ggtgctgcct cagattcagg ttatgccgct
     5881 caattcgctg cgtatatcgc ttgctgatta cgtgcagctt tcccttcagg cgggattcat
     5941 acagcggcca gccatccgtc atccatatca ccacgtcaaa gggtgacagc aggctcataa
     6001 gacgccccag cgtcgccata gtgcgttcac cgaatacgtg cgcaacaacc gtcttccgga
     6061 gactgtcata cgcgtaaaac agccagcgct ggcgcgattt agccccgaca tagccccact
     6121 gttcgtccat ttccgcgcag acgatgacgt cactgcccgg ctgtatgcgc gaggttaccg
     6181 actgcggcct gagtttttta agtgacgtaa aatcgtgttg aggccaacgc ccataatgcg
     6241 ggctgttgcc cggcatccaa cgccattcat ggccatatca atgattttct ggtgcgtacc
     6301 gggttgagaa gcggtgtaag tgaactgcag ttgccatgtt ttacggcagt gagagcagag
     6361 atagcgctga tgtccggcgg tgcttttgcc gttacgcacc accccgtcag tagctgaaca
     6421 ggagggacag ctgatagaaa cagaagccac tggagcacct caaaaacacc atcatacact
     6481 aaatcagtaa gttggcagca tcaccggttt tttgtgtgaa tcgatagtac taacatacgc
     6541 tctccatcaa aacaaaacga aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag
     6601 tgcaggtgcc agaacatttc tct
//
