LOCUS       sequence-442606-        4933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of FLAG-tagged human MAD2 (R133E/Q134A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4933)
  AUTHORS   Chuah YH, Tay EXY, Grinchuk OV, Yoon J, Feng J, Kannan S, Robert M, 
            Jakhar R, Liang Y, Lee BWL, Wang LC, Lim YT, Zhao T, Sobota RM, Lu 
            G, Low BC, Crasta KC, Verma CS, Lin Z, Ong DST
  TITLE     CAMK2D serves as a molecular scaffold for RNF8-MAD2 complex to 
            induce mitotic checkpoint in glioma.
  JOURNAL   Cell Death Differ. 2023 Aug;30(8):1973-1987. doi: 
            10.1038/s41418-023-01192-3. Epub 2023 Jul 19.
  PUBMED    37468549
REFERENCE   2  (bases 1 to 4933)
  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"; doi: 
            "10.1038/s41418-023-01192-3"; journalName: "Cell Death Differ"; 
            date: "2023-08"; volume: "30"; issue: "8"; pages: "1973-1987"
COMMENT     Sequence Label: pCMV-FLAG-MAD2-RQ
FEATURES             Location/Qualifiers
     source          1..4933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        100..403
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        404..607
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     558..578
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        653..671
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     regulatory      706..715
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             715..738
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1395..1411)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1396..1412)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1467..1486)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1468..1486)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1760..1881
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1797..1816)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1851..1870
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1888..2343)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2025..2046)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2237..2256
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2370..2474
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2476..2833
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2476..2667
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2499..2519)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2684..2819
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2868..3662
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2868..3662
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2922..2941)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3532..3551
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3850..3869)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3894..3941
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4270..4858
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4759..4778
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcccctgatt ctgtggataa ccgtattacc gccatgcatt agttattaat agtaatcaat
       61 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      121 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      181 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      241 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      301 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      361 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
      421 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
      481 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
      541 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      601 cagagctggt ttagtgaacc gtcagatccg ctagcgatta cgccaagctc gaaattaacc
      661 ctcactaaag ggaacaaaag ctggagctcc accgcggtgg cggccgccac catggattac
      721 aaggatgacg acgataagag cccgggcgga tcccccgggc tgcaggaatt catggcgctg
      781 cagctctccc gggagcaggg aatcaccctg cgcgggagcg ccgaaatcgt ggccgagttc
      841 ttctcattcg gcatcaacag cattttatat cagcgtggca tatatccatc tgaaaccttt
      901 actcgagtgc agaaatacgg actcaccttg cttgtaacta ctgatcttga gctcataaaa
      961 tacctaaata atgtggtgga acaactgaaa gattggttat acaagtgttc agttcagaaa
     1021 ctggttgtag ttatctcaaa tattgaaagt ggtgaggtcc tggaaagatg gcagtttgat
     1081 attgagtgtg acaagactgc aaaagatgac agtgcaccca gagaaaagtc tcagaaagct
     1141 atccaggatg aaatccgttc agtgatcgag gcgatcacag ctacggtgac atttctgcca
     1201 ctgttggaag tttcttgttc atttgatctg ctgatttata cagacaaaga tttggttgta
     1261 cctgaaaaat gggaagagtc gggaccacag tttattacca attctgagga agtccgcctt
     1321 cgttcattta ctactacaat ccacaaagta aatagcatgg tggcctacaa aattcctgtc
     1381 aatgacaagc ttatcgatac cgtcgacctc gagggggggc ccggtacctt aattaattaa
     1441 ggtaccaggt aagtgtaccc aattcgccct atagtgagtc gtattacaat tcactcgatc
     1501 gcccttccca acagttgcgc agcctgaatg gcgaatggag atccaatttt taagtgtata
     1561 atgtgttaaa ctactgattc taattgtttg tgtattttag attcacagtc ccaaggctca
     1621 tttcaggccc ctcagtcctc acagtctgtt catgatcata atcagccata ccacatttgt
     1681 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     1741 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     1801 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     1861 caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     1921 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     1981 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     2041 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     2101 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     2161 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     2221 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     2281 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     2341 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2401 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2461 gaaaaaggaa gaatcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     2521 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2581 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2641 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     2701 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     2761 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     2821 aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     2881 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     2941 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3001 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
     3061 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3121 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3181 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3241 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3301 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagaa catcaggggc
     3361 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
     3421 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     3481 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     3541 cccgtgatat tgctgaagaa cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     3601 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     3661 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     3721 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     3781 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg
     3841 gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat
     3901 aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     3961 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     4021 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     4081 cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt
     4141 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4201 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4261 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4321 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4381 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     4441 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4501 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4561 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4621 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4681 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4741 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4801 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4861 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4921 ctttcctgcg tta
//
