LOCUS       sequence-73909-e        4477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  minP-luc2 donor plasmid for MPRA library construction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4477)
  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 4477)
  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: pMPRAdonor2
FEATURES             Location/Qualifiers
     source          1..4477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        64..95
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      122..131
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             128..1780
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(1849..1865)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1849..1865)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1862..1884)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1873..1889
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1897..1903,1904..1921,1922..1927))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1942..1963
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2080..2097)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2251..2839)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2331..2350)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3010..3801,3802..3870))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3633..3652
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3871..3975)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4043..4061
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4099..4121)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4221..4240
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4434..4456
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4448..4465
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4449..4465
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ggtacctgag ctcgctagcc tcgaggatat caagatctgg cctcggcggc caagcttaga
       61 cactagaggg tatataatgg aagctcgact tccagcttgg caatccggta ctgttggtaa
      121 agccaccatg gaagatgcca aaaacattaa gaagggccca gcgccattct acccactcga
      181 agacgggacc gccggcgagc agctgcacaa agccatgaag cgctacgccc tggtgcccgg
      241 caccatcgcc tttaccgacg cacatatcga ggtggacatt acctacgccg agtacttcga
      301 gatgagcgtt cggctggcag aagctatgaa gcgctatggg ctgaatacaa accatcggat
      361 cgtggtgtgc agcgagaata gcttgcagtt cttcatgccc gtgttgggtg ccctgttcat
      421 cggtgtggct gtggccccag ctaacgacat ctacaacgag cgcgagctgc tgaacagcat
      481 gggcatcagc cagcccaccg tcgtattcgt gagcaagaaa gggctgcaaa agatcctcaa
      541 cgtgcaaaag aagctaccga tcatacaaaa gatcatcatc atggatagca agaccgacta
      601 ccagggcttc caaagcatgt acaccttcgt gacttcccat ttgccacccg gcttcaacga
      661 gtacgacttc gtgcccgaga gcttcgaccg ggacaaaacc atcgccctga tcatgaacag
      721 tagtggcagt accggattgc ccaagggcgt agccctaccg caccgcaccg cttgtgtccg
      781 attcagtcat gcccgcgacc ccatcttcgg caaccagatc atccccgaca ccgctatcct
      841 cagcgtggtg ccatttcacc acggcttcgg catgttcacc acgctgggct acttgatctg
      901 cggctttcgg gtcgtgctca tgtaccgctt cgaggaggag ctattcttgc gcagcttgca
      961 agactataag attcaatctg ccctgctggt gcccacacta tttagcttct tcgctaagag
     1021 cactctcatc gacaagtacg acctaagcaa cttgcacgag atcgccagcg gcggggcgcc
     1081 gctcagcaag gaggtaggtg aggccgtggc caaacgcttc cacctaccag gcatccgcca
     1141 gggctacggc ctgacagaaa caaccagcgc cattctgatc acccccgaag gggacgacaa
     1201 gcctggcgca gtaggcaagg tggtgccctt cttcgaggct aaggtggtgg acttggacac
     1261 cggtaagaca ctgggtgtga accagcgcgg cgagctgtgc gtccgtggcc ccatgatcat
     1321 gagcggctac gttaacaacc ccgaggctac aaacgctctc atcgacaagg acggctggct
     1381 gcacagcggc gacatcgcct actgggacga ggacgagcac ttcttcatcg tggaccggct
     1441 gaagagcctg atcaaataca agggctacca ggtagcccca gccgaactgg agagcatcct
     1501 gctgcaacac cccaacatct tcgacgccgg ggtcgccggc ctgcccgacg acgatgccgg
     1561 cgagctgccc gccgcagtcg tcgtgctgga acacggtaaa accatgaccg agaaggagat
     1621 cgtggactat gtggccagcc aggttacaac cgccaagaag ctgcgcggtg gtgttgtgtt
     1681 cgtggacgag gtgcctaaag gactgaccgg caagttggac gcccgcaaga tccgcgagat
     1741 tctcattaag gccaagaagg gcggcaagat cgccgtgtaa taattctaga tatcggatcc
     1801 cgggcccgtc gactgcagag gcctgcatgc aagcttggcg taatcatggt catagctgtt
     1861 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     1921 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     1981 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     2041 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     2101 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     2161 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     2221 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2281 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2341 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2401 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     2461 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     2521 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     2581 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     2641 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     2701 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     2761 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     2821 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     2881 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     2941 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3001 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3061 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3121 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     3181 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3241 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3301 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3361 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3421 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     3481 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     3541 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     3601 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     3661 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     3721 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     3781 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     3841 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     3901 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     3961 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     4021 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     4081 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     4141 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     4201 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     4261 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt
     4321 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     4381 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     4441 acgacgttgt aaaacgacgg ccagtgaatt cgagctc
//
