LOCUS       sequence-329146-        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"
     intron          120..252
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     235..254
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        297..315
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      319..328
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             325..1260
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     promoter        1366..2117
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     regulatory      2147..2156
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2153..3805
                     /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             3112..3120
                     /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    3849..3970
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3860..3879)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3914..3933
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4103..4207
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4208..4276,4277..5068)
                     /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(4426..4445)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5239..5827
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5728..5747
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(5956..6263,1..50)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5956..6147
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5979..5999)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(6164..6263,1..36)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagcttgatt
       61 cttctgacac aacagtctcg aacttaagct gcagaagttg gtcgtgaggc actgggcagg
      121 taagtatcaa ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac
      181 agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct
      241 ttctctccac aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat
      301 acgactcact ataggctagc caccatggct tccaaggtgt acgaccccga gcaacgcaaa
      361 cgcatgatca ctgggcctca gtggtgggct cgctgcaagc aaatgaacgt gctggactcc
      421 ttcatcaact actatgattc cgagaagcac gccgagaacg ccgtgatttt tctgcatggt
      481 aacgctgcct ccagctacct gtggaggcac gtcgtgcctc acatcgagcc cgtggctaga
      541 tgcatcatcc ctgatctgat cggaatgggt aagtccggca agagcgggaa tggctcatat
      601 cgcctcctgg atcactacaa gtacctcacc gcttggttcg agctgctgaa ccttccaaag
      661 aaaatcatct ttgtgggcca cgactggggg gcttgtctgg cctttcacta ctcctacgag
      721 caccaagaca agatcaaggc catcgtccat gctgagagtg tcgtggacgt gatcgagtcc
      781 tgggacgagt ggcctgacat cgaggaggat atcgccctga tcaagagcga agagggcgag
      841 aaaatggtgc ttgagaataa cttcttcgtc gagaccatgc tcccaagcaa gatcatgcgg
      901 aaactggagc ctgaggagtt cgctgcctac ctggagccat tcaaggagaa gggcgaggtt
      961 agacggccta ccctctcctg gcctcgcgag atccctctcg ttaagggagg caagcccgac
     1021 gtcgtccaga ttgtccgcaa ctacaacgcc taccttcggg ccagcgacga tctgcctaag
     1081 atgttcatcg agtccgaccc tgggttcttt tccaacgcta ttgtcgaggg agctaagaag
     1141 ttccctaaca ccgagttcgt gaaggtgaag ggcctccact tcagccagga ggacgctcca
     1201 gatgaaatgg gtaagtacat caagagcttc gtggagcgcg tgctgaagaa cgagcagtaa
     1261 ttctaggcga tcgctcgagc ccgggaattc gtttaaacct agagcggccg ctggccgcaa
     1321 aaaaaaaccc cgcccctgac agggcggggt tttttttagg atctaaatga gtcttcggac
     1381 ctcgcggggg ccgcttaagc ggtggttagg gtttgtctga cgcgggggga gggggaagga
     1441 acgaaacact ctcattcgga ggcggctcgg ggtttggtct tggtggccac gggcacgcag
     1501 aagagcgccg cgatcctctt aagcaccccc ccgccctccg tggaggcggg ggtttggtcg
     1561 gcgggtggta actggcgggc cgctgactcg ggcgggtcgc gcgccccaga gtgtgacctt
     1621 ttcggtctgc tcgcagaccc ccgggcggcg ccgccgcggc ggcgacgggc tcgctgggtc
     1681 ctaggctcca tggggaccgt atacgtggac aggctctgga gcatccgcac gactgcggtg
     1741 atattaccgg agaccttctg cgggacgagc cgggtcacgc ggctgacgcg gagcgtccgt
     1801 tgggcgacaa acaccaggac ggggcacagg tacactatct tgtcacccgg aggcgcgagg
     1861 gactgcagga gcttcaggga gtggcgcagc tgcttcatcc ccgtggcccg ttgctcgcgt
     1921 ttgctggcgg tgtccccgga agaaatatat ttgcatgtct ttagttctat gatgacacaa
     1981 accccgccca gcgtcttgtc attggcgaat tcgaacacgc agatgcagtc ggggcggcgc
     2041 ggtcccaggt ccacttcgca tattaaggtg acgcgtgtgg cctcgaacac cgagcgaccc
     2101 tgcagcgacc cgcttaaaag cttggcattc cggtactgtt ggtaaagcca ccatggccga
     2161 tgctaagaac attaagaagg gccctgctcc cttctaccct ctggaggatg gcaccgctgg
     2221 cgagcagctg cacaaggcca tgaagaggta tgccctggtg cctggcacca ttgccttcac
     2281 cgatgcccac attgaggtgg acatcaccta tgccgagtac ttcgagatgt ctgtgcgcct
     2341 ggccgaggcc atgaagaggt acggcctgaa caccaaccac cgcatcgtgg tgtgctctga
     2401 gaactctctg cagttcttca tgccagtgct gggcgccctg ttcatcggag tggccgtggc
     2461 ccctgctaac gacatttaca acgagcgcga gctgctgaac agcatgggca tttctcagcc
     2521 taccgtggtg ttcgtgtcta agaagggcct gcagaagatc ctgaacgtgc agaagaagct
     2581 gcctatcatc cagaagatca tcatcatgga ctctaagacc gactaccagg gcttccagag
     2641 catgtacaca ttcgtgacat ctcatctgcc tcctggcttc aacgagtacg acttcgtgcc
     2701 agagtctttc gacagggaca aaaccattgc cctgatcatg aacagctctg ggtctaccgg
     2761 cctgcctaag ggcgtggccc tgcctcatcg caccgcctgt gtgcgcttct ctcacgcccg
     2821 cgaccctatt ttcggcaacc agatcatccc cgacaccgct attctgagcg tggtgccatt
     2881 ccaccacggc ttcggcatgt tcaccaccct gggctacctg atttgcggct ttcgggtggt
     2941 gctgatgtac cgcttcgagg aggagctgtt cctgcgcagc ctgcaagact acaaaattca
     3001 gtctgccctg ctggtgccaa ccctgttcag cttcttcgct aagagcaccc tgatcgacaa
     3061 gtacgacctg tctaacctgc acgagattgc ctctggcggc gccccactgt ctaaggaggt
     3121 gggcgaagcc gtggccaagc gctttcatct gccaggcatc cgccagggct acggcctgac
     3181 cgagacaacc agcgccattc tgattacccc agagggcgac gacaagcctg gcgccgtggg
     3241 caaggtggtg ccattcttcg aggccaaggt ggtggacctg gacaccggca agaccctggg
     3301 agtgaaccag cgcggcgagc tgtgtgtgcg cggccctatg attatgtccg gctacgtgaa
     3361 taaccctgag gccacaaacg ccctgatcga caaggacggc tggctgcact ctggcgacat
     3421 tgcctactgg gacgaggacg agcacttctt catcgtggac cgcctgaagt ctctgatcaa
     3481 gtacaagggc taccaggtgg ccccagccga gctggagtct atcctgctgc agcaccctaa
     3541 cattttcgac gccggagtgg ccggcctgcc cgacgacgat gccggcgagc tgcctgccgc
     3601 cgtcgtcgtg ctggaacacg gcaagaccat gaccgagaag gagatcgtgg actatgtggc
     3661 cagccaggtg acaaccgcca agaagctgcg cggcggagtg gtgttcgtgg acgaggtgcc
     3721 caagggcctg accggcaagc tggacgcccg caagatccgc gagatcctga tcaaggctaa
     3781 gaaaggcggc aagatcgccg tgtaataatt ctagagtcgg ggcggccggc cgcttcgagc
     3841 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     3901 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     3961 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     4021 ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatcgata aggatccagg
     4081 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4141 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4201 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4261 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     4321 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4381 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4441 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4501 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4561 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4621 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4681 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4741 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4801 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4861 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4921 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4981 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5041 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5101 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5161 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5221 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5281 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5341 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5401 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5461 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5521 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5581 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5641 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5701 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5761 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5821 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5881 tcacatggct cgacagatct gcgcagcacc atggcctgaa ataacctctg aaagaggaac
     5941 ttggttaggt accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     6001 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     6061 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     6121 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     6181 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     6241 cgaggccgcc tcggcctctg agc
//
