LOCUS       sequence-83433-e        5597 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter control for pMPRA2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5597)
  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 5597)
  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-CMV-Clover
FEATURES             Location/Qualifiers
     source          1..5597
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..392
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        393..596
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     547..567
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     641..660
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        641..659
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(725..727,728..730,731..1441)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(770..791)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1378..1399
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1457..1475)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1458..1475)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     polyA_signal    1533..1654
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1544..1563)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1598..1617
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        1756..2256
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2175..2193
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      2257..2266
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3309..3357
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(3524..3541)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3695..4283)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3775..3794)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4484..5275,5276..5344))
                     /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    5444..5492
                     /note="synthetic polyadenylation signal"
     misc_feature    5506..5597
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5546..5565
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggcctaactg gccacattga ttattgacta gttattaata gtaatcaatt acggggtcat
       61 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      121 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      181 cgccaatagg gactttccat tgacgtcaat gggtggacta tttacggtaa actgcccact
      241 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      301 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      361 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      421 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      481 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      541 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct
      601 ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact cactataggg
      661 agacccaagc ttggtaccga gctcggatcc actagtaacg gccgccagtg tgctggaatt
      721 cggcatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      781 ggacggcgac gtaaacggcc acaagttcag cgtccgcggc gagggcgagg gcgatgccac
      841 caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      901 caccctcgtg accaccttcg gctacggcgt ggcctgcttc agccgctacc ccgaccacat
      961 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1021 ctctttcaag gacgacggta cctacaagac ccgcgccgag gtgaagttcg agggcgacac
     1081 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1141 gcacaagctg gagtacaact tcaacagcca caacgtctat atcacggccg acaagcagaa
     1201 gaacggcatc aaggctaact tcaagatccg ccacaacgtt gaggacggca gcgtgcagct
     1261 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1321 ccactacctg agccatcagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1381 ggtcctgctg gagttcgtga ccgccgccgg gattacacat ggcatggacg agctgtacaa
     1441 gtctagaggg ccctattcta tagtgtcacc taaatgctag ggcctcggcg gccaagctag
     1501 tcggggcggc cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac
     1561 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     1621 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     1681 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     1741 tggtaaaatc gataattgcg ccttttccaa ggcagccctg ggtttgcgca gggacgcggc
     1801 tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc ctgggtctcg cacattcttc
     1861 acgtccgttc gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc cccggcgacg
     1921 cttcctgctc cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga
     1981 caaacggaag ccgcacgtct cactagtacc ctcgcagacg gacagcgcca gggagcaatg
     2041 gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct gctcagcagg gcgcgccgag
     2101 agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg
     2161 ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc
     2221 tccctcgttg accgaatcac cgacctctct ccccaggcca ccatggcacc ctgcatgctg
     2281 ctcctgctgt tggcggctgc cctggccccg actcagaccc gcgcgggccc acattcgctg
     2341 aggtatttcc acaccgccgt gtcccggccc ggcctcggga agccccggtt catctctgtc
     2401 ggctacgtgg acgacacgca gttcgtgcgc ttcgacagcg acgcggagaa tccgaggtat
     2461 gagccgcggg tgcggtggat ggagcaggtg gagcccgagt attgggagcg gaacacgcag
     2521 atcgccaagg gcaatgagca gattttccga gtgaacctga ggaccgcgct gcgctactac
     2581 aaccagagcg cgggcggctc tcacacgttc caacggatgt acggctgtga ggtggggtcg
     2641 gactggcgcc tcctccgcgg gtacgagcag tacgcatacg acggctgcga ttacatcgcc
     2701 ctgaacgaag acctgaaaac gtggacggcg gccgacatgg cggcgctgat caccaaacac
     2761 aagtgggagc aggctggtga tgcagagaga gaccgggcct acctggaggg cacgtgcgtg
     2821 gagtggctcc gcagatacct gcagctcggg aacgcgacgc tgccgcgcac agattcccca
     2881 aaggcccatg tgacccgtca cagcagacct gaagataaag tcaccctgag gtgctgggcc
     2941 ctgggcttct accctgctga catcaccctg acctggcagt tgaatgggga ggagctgacc
     3001 caggacatgg agcttgtgga gaccaggcct gcaggggatg gaaccttcca gaagtgggca
     3061 tctgtggtgg tgcctcttgg gaaggagcag tattacacat gccatgtgta ccatcagggg
     3121 ctgcctgagc ccctcaccct gagatgggag cctcctccat ccactgtctc caacacggta
     3181 atcattgctg ttctggttgt ccttggagct gcaatagtca ctggagctgt ggtggctttt
     3241 gtgatgaaga tgagaaggag aaacacaggt ggaaaaggag ggtgaactct tgaaccggtc
     3301 atggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtggga
     3361 tccgtcgacc gatgcccttg agagccttca acccagtcag ctccttttca acccagtcag
     3421 ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat
     3481 catgcaactc gtaggacagg tgccggcagc gctcttccgc ttcctcgctc actgactcgc
     3541 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3601 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3661 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3721 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3781 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3841 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3901 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3961 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4021 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4081 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4141 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4201 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4261 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4321 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4381 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4441 cttggtctga cagcggccgc aaatgctaaa ccactgcagt ggtttaccag tgcttgatca
     4501 gtgaggcacc gatctcagcg atctgcctat ttcgttcgtc catagtggcc tgactccccg
     4561 tcgtgtagat cactacgatt cgtgagggct taccatcagg ccccagcgca gcaatgatgc
     4621 cgcgagagcc gcgttcaccg gcccccgatt tgtcagcaat gaaccagcca gcagggaggg
     4681 ccgagcgaag aagtggtcct gctactttgt ccgcctccat ccagtctatg agctgctgtc
     4741 gtgatgctag agtaagaagt tcgccagtga gtagtttccg aagagttgtg gccattgcta
     4801 ctggcatcgt ggtatcacgc tcgtcgttcg gtatggcttc gttcaactct ggttcccagc
     4861 ggtcaagccg ggtcacatga tcacccatat tatgaagaaa tgcagtcagc tccttagggc
     4921 ctccgatcgt tgtcagaagt aagttggccg cggtgttgtc gctcatggta atggcagcac
     4981 tacacaattc tcttaccgtc atgccatccg taagatgctt ttccgtgacc ggcgagtact
     5041 caaccaagtc gttttgtgag tagtgtatac ggcgaccaag ctgctcttgc ccggcgtcta
     5101 tacgggacaa caccgcgcca catagcagta ctttgaaagt gctcatcatc gggaatcgtt
     5161 cttcggggcg gaaagactca aggatcttgc cgctattgag atccagttcg atatagccca
     5221 ctcttgcacc cagttgatct tcagcatctt ttactttcac cagcgtttcg gggtgtgcaa
     5281 aaacaggcaa gcaaaatgcc gcaaagaagg gaatgagtgc gacacgaaaa tgttggatgc
     5341 tcattccttt ttcaatatta ttgaagcatt tatcagggtt actagtacgt ctctcaagga
     5401 taagtaagta atattaaggt acgggaggta ttggacaggc cgcaataaaa tatctttatt
     5461 ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gtactaacat acgctctcca
     5521 tcaaaacaaa acgaaacaaa acaaactagc aaaataggct gtccccagtg caagtgcagg
     5581 tgccagaaca tttctct
//
