LOCUS       sequence-325511-        6263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The mpCHECK2 plasmid has the ability to evaluate the effect of 
            polyadenylation signal variants on expression of disease-associated 
            genes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6263)
  AUTHORS   Chen M, Wei R, Wei G, Xu M, Su Z, Zhao C, Ni T
  TITLE     Systematic evaluation of the effect of polyadenylation signal 
            variants on the expression of disease-associated genes.
  JOURNAL   Genome Res. 2021 Apr 19. pii: gr.270256.120. doi: 
            10.1101/gr.270256.120.
  PUBMED    33875481
REFERENCE   2  (bases 1 to 6263)
  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: "Genome Res.
            2021 Apr 19. pii: gr.270256.120. doi: 10.1101/gr.270256.120."
COMMENT     Sequence Label: mpCHECK2
FEATURES             Location/Qualifiers
     source          1..6263
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        62..419
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        62..253
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(85..105)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      270..405
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          489..621
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     604..623
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        666..684
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      688..697
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             694..1629
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     promoter        1735..2486
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     regulatory      2516..2525
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2522..4174
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /db_xref="GI:42718124"
                     /protein_id="AAS38485.1"
                     /translation="MADAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     RBS             3481..3489
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    4218..4339
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4229..4248)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4283..4302
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4472..4576
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4577..4645,4646..5437)
                     /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     complement(4795..4814)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5608..6196
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6097..6116
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct
       61 tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
      121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
      181 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
      241 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
      301 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
      361 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
      421 agcttgattc ttctgacaca acagtctcga acttaagctg cagaagttgg tcgtgaggca
      481 ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct
      541 tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc
      601 actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga
      661 gtacttaata cgactcacta taggctagcc accatggctt ccaaggtgta cgaccccgag
      721 caacgcaaac gcatgatcac tgggcctcag tggtgggctc gctgcaagca aatgaacgtg
      781 ctggactcct tcatcaacta ctatgattcc gagaagcacg ccgagaacgc cgtgattttt
      841 ctgcatggta acgctgcctc cagctacctg tggaggcacg tcgtgcctca catcgagccc
      901 gtggctagat gcatcatccc tgatctgatc ggaatgggta agtccggcaa gagcgggaat
      961 ggctcatatc gcctcctgga tcactacaag tacctcaccg cttggttcga gctgctgaac
     1021 cttccaaaga aaatcatctt tgtgggccac gactgggggg cttgtctggc ctttcactac
     1081 tcctacgagc accaagacaa gatcaaggcc atcgtccatg ctgagagtgt cgtggacgtg
     1141 atcgagtcct gggacgagtg gcctgacatc gaggaggata tcgccctgat caagagcgaa
     1201 gagggcgaga aaatggtgct tgagaataac ttcttcgtcg agaccatgct cccaagcaag
     1261 atcatgcgga aactggagcc tgaggagttc gctgcctacc tggagccatt caaggagaag
     1321 ggcgaggtta gacggcctac cctctcctgg cctcgcgaga tccctctcgt taagggaggc
     1381 aagcccgacg tcgtccagat tgtccgcaac tacaacgcct accttcgggc cagcgacgat
     1441 ctgcctaaga tgttcatcga gtccgaccct gggttctttt ccaacgctat tgtcgaggga
     1501 gctaagaagt tccctaacac cgagttcgtg aaggtgaagg gcctccactt cagccaggag
     1561 gacgctccag atgaaatggg taagtacatc aagagcttcg tggagcgcgt gctgaagaac
     1621 gagcagtaat tctaggcgat cgctcgagcc cgggaattcg tttaaaccta gagcggccgc
     1681 tggccgcaaa aaaaaacccc gcccctgaca gggcggggtt ttttttagga tctaaatgag
     1741 tcttcggacc tcgcgggggc cgcttaagcg gtggttaggg tttgtctgac gcggggggag
     1801 ggggaaggaa cgaaacactc tcattcggag gcggctcggg gtttggtctt ggtggccacg
     1861 ggcacgcaga agagcgccgc gatcctctta agcacccccc cgccctccgt ggaggcgggg
     1921 gtttggtcgg cgggtggtaa ctggcgggcc gctgactcgg gcgggtcgcg cgccccagag
     1981 tgtgaccttt tcggtctgct cgcagacccc cgggcggcgc cgccgcggcg gcgacgggct
     2041 cgctgggtcc taggctccat ggggaccgta tacgtggaca ggctctggag catccgcacg
     2101 actgcggtga tattaccgga gaccttctgc gggacgagcc gggtcacgcg gctgacgcgg
     2161 agcgtccgtt gggcgacaaa caccaggacg gggcacaggt acactatctt gtcacccgga
     2221 ggcgcgaggg actgcaggag cttcagggag tggcgcagct gcttcatccc cgtggcccgt
     2281 tgctcgcgtt tgctggcggt gtccccggaa gaaatatatt tgcatgtctt tagttctatg
     2341 atgacacaaa ccccgcccag cgtcttgtca ttggcgaatt cgaacacgca gatgcagtcg
     2401 gggcggcgcg gtcccaggtc cacttcgcat attaaggtga cgcgtgtggc ctcgaacacc
     2461 gagcgaccct gcagcgaccc gcttaaaagc ttggcattcc ggtactgttg gtaaagccac
     2521 catggccgat gctaagaaca ttaagaaggg ccctgctccc ttctaccctc tggaggatgg
     2581 caccgctggc gagcagctgc acaaggccat gaagaggtat gccctggtgc ctggcaccat
     2641 tgccttcacc gatgcccaca ttgaggtgga catcacctat gccgagtact tcgagatgtc
     2701 tgtgcgcctg gccgaggcca tgaagaggta cggcctgaac accaaccacc gcatcgtggt
     2761 gtgctctgag aactctctgc agttcttcat gccagtgctg ggcgccctgt tcatcggagt
     2821 ggccgtggcc cctgctaacg acatttacaa cgagcgcgag ctgctgaaca gcatgggcat
     2881 ttctcagcct accgtggtgt tcgtgtctaa gaagggcctg cagaagatcc tgaacgtgca
     2941 gaagaagctg cctatcatcc agaagatcat catcatggac tctaagaccg actaccaggg
     3001 cttccagagc atgtacacat tcgtgacatc tcatctgcct cctggcttca acgagtacga
     3061 cttcgtgcca gagtctttcg acagggacaa aaccattgcc ctgatcatga acagctctgg
     3121 gtctaccggc ctgcctaagg gcgtggccct gcctcatcgc accgcctgtg tgcgcttctc
     3181 tcacgcccgc gaccctattt tcggcaacca gatcatcccc gacaccgcta ttctgagcgt
     3241 ggtgccattc caccacggct tcggcatgtt caccaccctg ggctacctga tttgcggctt
     3301 tcgggtggtg ctgatgtacc gcttcgagga ggagctgttc ctgcgcagcc tgcaagacta
     3361 caaaattcag tctgccctgc tggtgccaac cctgttcagc ttcttcgcta agagcaccct
     3421 gatcgacaag tacgacctgt ctaacctgca cgagattgcc tctggcggcg ccccactgtc
     3481 taaggaggtg ggcgaagccg tggccaagcg ctttcatctg ccaggcatcc gccagggcta
     3541 cggcctgacc gagacaacca gcgccattct gattacccca gagggcgacg acaagcctgg
     3601 cgccgtgggc aaggtggtgc cattcttcga ggccaaggtg gtggacctgg acaccggcaa
     3661 gaccctggga gtgaaccagc gcggcgagct gtgtgtgcgc ggccctatga ttatgtccgg
     3721 ctacgtgaat aaccctgagg ccacaaacgc cctgatcgac aaggacggct ggctgcactc
     3781 tggcgacatt gcctactggg acgaggacga gcacttcttc atcgtggacc gcctgaagtc
     3841 tctgatcaag tacaagggct accaggtggc cccagccgag ctggagtcta tcctgctgca
     3901 gcaccctaac attttcgacg ccggagtggc cggcctgccc gacgacgatg ccggcgagct
     3961 gcctgccgcc gtcgtcgtgc tggaacacgg caagaccatg accgagaagg agatcgtgga
     4021 ctatgtggcc agccaggtga caaccgccaa gaagctgcgc ggcggagtgg tgttcgtgga
     4081 cgaggtgccc aagggcctga ccggcaagct ggacgcccgc aagatccgcg agatcctgat
     4141 caaggctaag aaaggcggca agatcgccgt gtaataattc tagagtcggg gcggccggcc
     4201 gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     4261 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     4321 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     4381 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatcgataa
     4441 ggatccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4501 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4561 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4621 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     4681 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4741 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4801 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4861 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     4921 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     4981 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5041 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     5101 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     5161 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     5221 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     5281 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     5341 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5401 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5461 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5521 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5581 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5641 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5701 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5761 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5821 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5881 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5941 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6001 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6061 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6121 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6181 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6241 gccttttgct cacatggctc gac
//
