LOCUS       sequence-450110-        6559 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for transient transfection of Massively Parallel Reporter 
            Assay (MPRA) libraries, pSV40-H2Kk for physical selection of 
            transfected cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6559)
  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 6559)
  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: pMPRA3
FEATURES             Location/Qualifiers
     source          1..6559
                     /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        2076..2433
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2076..2267
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2099..2119)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2284..2419
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2346..2365
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     regulatory      2458..2467
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3510..3558
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(3705..3722)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3876..4464)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3956..3975)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4664..5455,5456..5524))
                     /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  5674..6441
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     misc_feature    6468..6559
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     6508..6527
                     /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 gcgcagcacc atggcctgaa
     2041 ataacctctg aaagaggaac ttggttagct accttctgag gcggaaagaa ccagctgtgg
     2101 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2161 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     2221 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     2281 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     2341 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     2401 ggaggctttt ttggaggcct aggcttttgc aaaaagctcg attcttctga cactagcgcc
     2461 accatggcac cctgcatgct gctcctgctg ttggcggctg ccctggcccc gactcagacc
     2521 cgcgcgggcc cacattcgct gaggtatttc cacaccgccg tgtcccggcc cggcctcggg
     2581 aagccccggt tcatctctgt cggctacgtg gacgacacgc agttcgtgcg cttcgacagc
     2641 gacgcggaga atccgaggta tgagccgcgg gtgcggtgga tggagcaggt ggagcccgag
     2701 tattgggagc ggaacacgca gatcgccaag ggcaatgagc agattttccg agtgaacctg
     2761 aggaccgcgc tgcgctacta caaccagagc gcgggcggct ctcacacgtt ccaacggatg
     2821 tacggctgtg aggtggggtc ggactggcgc ctcctccgcg ggtacgagca gtacgcatac
     2881 gacggctgcg attacatcgc cctgaacgaa gacctgaaaa cgtggacggc ggccgacatg
     2941 gcggcgctga tcaccaaaca caagtgggag caggctggtg atgcagagag agaccgggcc
     3001 tacctggagg gcacgtgcgt ggagtggctc cgcagatacc tgcagctcgg gaacgcgacg
     3061 ctgccgcgca cagattcccc aaaggcccat gtgacccgtc acagcagacc tgaagataaa
     3121 gtcaccctga ggtgctgggc cctgggcttc taccctgctg acatcaccct gacctggcag
     3181 ttgaatgggg aggagctgac ccaggacatg gagcttgtgg agaccaggcc tgcaggggat
     3241 ggaaccttcc agaagtgggc atctgtggtg gtgcctcttg ggaaggagca gtattacaca
     3301 tgccatgtgt accatcaggg gctgcctgag cccctcaccc tgagatggga gcctcctcca
     3361 tccactgtct ccaacacggt aatcattgct gttctggttg tccttggagc tgcaatagtc
     3421 actggagctg tggtggcttt tgtgatgaag atgagaagga gaaacacagg tggaaaagga
     3481 gggtgaactc ttgaaccggt catggccgca ataaaatatc tttattttca ttacatctgt
     3541 gtgttggttt tttgtgtggg atccgtcgac cgatgccctt gagagccttc aacccagtca
     3601 gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
     3661 tcatgcaact cgtaggacag gtgccggcag cgctcttccg cttcctcgct cactgactcg
     3721 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3781 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3841 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3901 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3961 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4021 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4081 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4141 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4201 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4261 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4321 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4381 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4441 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4501 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4561 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4621 acttggtctg acagcggccg caaatgctaa accactgcag tggttaccag tgcttgatca
     4681 gtgaggcacc gatctcagcg atctgcctat ttcgttcgtc catagtggcc tgactccccg
     4741 tcgtgtagat cactacgatt cgtgagggct taccatcagg ccccagcgca gcaatgatgc
     4801 cgcgagagcc gcgttcaccg gcccccgatt tgtcagcaat gaaccagcca gcagggaggg
     4861 ccgagcgaag aagtggtcct gctactttgt ccgcctccat ccagtctatg agctgctgtc
     4921 gtgatgctag agtaagaagt tcgccagtga gtagtttccg aagagttgtg gccattgcta
     4981 ctggcatcgt ggtatcacgc tcgtcgttcg gtatggcttc gttcaactct ggttcccagc
     5041 ggtcaagccg ggtcacatga tcacccatat tatgaagaaa tgcagtcagc tccttagggc
     5101 ctccgatcgt tgtcagaagt aagttggccg cggtgttgtc gctcatggta atggcagcac
     5161 tacacaattc tcttaccgtc atgccatccg taagatgctt ttccgtgacc ggcgagtact
     5221 caaccaagtc gttttgtgag tagtgtatac ggcgaccaag ctgctcttgc ccggcgtcta
     5281 tacgggacaa caccgcgcca catagcagta ctttgaaagt gctcatcatc gggaatcgtt
     5341 cttcggggcg gaaagactca aggatcttgc cgctattgag atccagttcg atatagccca
     5401 ctcttgcacc cagttgatct tcagcatctt ttactttcac cagcgtttcg gggtgtgcaa
     5461 aaacaggcaa gcaaaatgcc gcaaagaagg gaatgagtgc gacacgaaaa tgttggatgc
     5521 tcatactcgt cctttttcaa tattattgaa gcatttatca gggttactag tacgtctctc
     5581 aaggataagt aagtaatatt aaggtacggg aggtattgga caggccgcaa taaaatatct
     5641 ttattttcat tacatctgtg tgttggtttt ttgggtaatg actccaactt attgatagtg
     5701 ttttatgttc agataatgcc cgatgacttt gtcatgcagc tccaccgatt ttgagaacga
     5761 cagcgacttc cgtcccagcc gtgccaggtg ctgcctcaga ttcaggttat gccgctcaat
     5821 tcgctgcgta tatcgcttgc tgattacgtg cagctttccc ttcaggcggg attcatacag
     5881 cggccagcca tccgtcatcc atatcaccac gtcaaagggt gacagcaggc tcataagacg
     5941 ccccagcgtc gccatagtgc gttcaccgaa tacgtgcgca acaaccgtct tccggagact
     6001 gtcatacgcg taaaacagcc agcgctggcg cgatttagcc ccgacatagc cccactgttc
     6061 gtccatttcc gcgcagacga tgacgtcact gcccggctgt atgcgcgagg ttaccgactg
     6121 cggcctgagt tttttaagtg acgtaaaatc gtgttgaggc caacgcccat aatgcgggct
     6181 gttgcccggc atccaacgcc attcatggcc atatcaatga ttttctggtg cgtaccgggt
     6241 tgagaagcgg tgtaagtgaa ctgcagttgc catgttttac ggcagtgaga gcagagatag
     6301 cgctgatgtc cggcggtgct tttgccgtta cgcaccaccc cgtcagtagc tgaacaggag
     6361 ggacagctga tagaaacaga agccactgga gcacctcaaa aacaccatca tacactaaat
     6421 cagtaagttg gcagcatcac cggttttttg tgtgaatcga tagtactaac atacgctctc
     6481 catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca
     6541 ggtgccagaa catttctct
//
