LOCUS       sequence-471812-        6333 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express TP53(Y220C)-HaloTag(R) Fusion Protein in Mammalian Cells 
            under a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6333)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6333)
  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: 
            "Unpublished"
COMMENT     Sequence Label: TP53(Y220C)-HaloTag Fusion Vector
FEATURES             Location/Qualifiers
     source          1..6333
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    3..84
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        317..620
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        621..824
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     775..795
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          960..1092
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1075..1094
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1136..1155
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1136..1154
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1168..2346
                     /codon_start=1
                     /gene="Homo sapiens TP53"
                     /product="human tumor suppressor protein that is often 
                     mutated in cancers"
                     /label=p53
                     /note="responds to cellular stresses to regulate the 
                     expression of target genes"
                     /translation="MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLML
                     SPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQ
                     GSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIY
                     KQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPCEP
                     PEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRD
                     RRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEM
                     FRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD"
     CDS             2368..2388
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             2404..3288
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     polyA_signal    3375..3496
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3386..3405)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3611..3644)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3654..3701
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3733..4527
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3787..3806)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4397..4416
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4685..5273)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4765..4784)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5399..5447
                     /note="synthetic polyadenylation signal"
     CDS             complement(5471..5869)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5866..5875
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5900..6257)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5914..6049
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6066..6257
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6214..6234
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 cggcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
       61 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
      121 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct
      181 gattatgatc cgcggccgca atacgtcgac gttatcagct gacttcgtac gagagcctag
      241 gattatggcg cgccactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      301 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      361 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      421 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      481 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      541 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      601 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      661 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      721 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      781 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      841 agatcactag aagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg
      901 cttctgacac aacagtctcg aacttaagct gcagaagttg gtcgtgaggc actgggcagg
      961 taagtatcaa ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac
     1021 agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct
     1081 ttctctccac aggtgtccac tcccagttca attacagctc ttaaggctag agtattaata
     1141 cgactcacta tagggctagc gatcgccatg gaggagccgc agtcagatcc tagcgtcgag
     1201 ccccctctga gtcaggaaac attttcagac ctatggaaac tacttcctga aaacaacgtt
     1261 ctgtccccct tgccgtccca agcaatggat gatttgatgc tgtccccgga cgatattgaa
     1321 caatggttca ctgaagaccc aggtccagat gaagctccca gaatgccaga ggctgctccc
     1381 cccgtggccc ctgcaccagc agctcctaca ccggcggccc ctgcaccagc cccctcctgg
     1441 cccctgtcat cttctgtccc ttcccagaaa acctaccagg gcagctacgg tttccgtctg
     1501 ggcttcttgc attctgggac agccaagtct gtgacttgca cgtactcccc tgccctcaac
     1561 aagatgtttt gccaactggc caagacctgc cctgtgcagc tgtgggttga ttccacaccc
     1621 ccgcccggca cccgcgtccg cgccatggcc atctacaagc agtcacagca catgacggag
     1681 gttgtgaggc gctgccccca ccatgagcgc tgctcagata gcgatggtct ggcccctcct
     1741 cagcatctta tccgagtgga aggaaatttg cgtgtggagt atttggatga cagaaacact
     1801 tttcgacata gtgtggtggt gccctgtgag ccgcctgagg ttggctctga ctgtaccacc
     1861 atccactaca actacatgtg taacagttcc tgcatgggcg gcatgaaccg gaggcccatc
     1921 ctcaccatca tcacactgga agactccagt ggtaatctac tgggacggaa cagctttgag
     1981 gtgcgtgttt gtgcctgtcc tgggagagac cggcgcacag aggaagagaa tctccgcaag
     2041 aaaggggagc ctcaccacga gctgccccca gggagcacta agcgagcact gcccaacaac
     2101 accagctcct ctccccagcc aaagaagaaa ccactggatg gagaatattt cacccttcag
     2161 atccgtgggc gtgagcgctt cgagatgttc cgagagctga atgaggcctt ggaactcaag
     2221 gatgcccagg ctgggaagga gccagggggg agcagggctc actccagcca cctgaagtcc
     2281 aaaaagggtc agtctacctc ccgccataaa aaactcatgt tcaagacaga agggcctgac
     2341 tcagacgttt ctctcgagcc aaccactgag gatctgtact ttcagagcga taacgatgga
     2401 tccgaaatcg gtactggctt tccattcgac ccccattatg tggaagtcct gggcgagcgc
     2461 atgcactacg tcgatgttgg tccgcgcgat ggcacccctg tgctgttcct gcacggtaac
     2521 ccgacctcct cctacgtgtg gcgcaacatc atcccgcatg ttgcaccgac ccatcgctgc
     2581 attgctccag acctgatcgg tatgggcaaa tccgacaaac cagacctggg ttatttcttc
     2641 gacgaccacg tccgcttcat ggatgccttc atcgaagccc tgggtctgga agaggtcgtc
     2701 ctggtcattc acgactgggg ctccgctctg ggtttccact gggccaagcg caatccagag
     2761 cgcgtcaaag gtattgcatt tatggagttc atccgcccta tcccgacctg ggacgaatgg
     2821 ccagaatttg cccgcgagac cttccaggcc ttccgcacca ccgacgtcgg ccgcaagctg
     2881 atcatcgatc agaacgtttt tatcgagggt acgctgccga tgggtgtcgt ccgcccgctg
     2941 actgaagtcg agatggacca ttaccgcgag ccgttcctga atcctgttga ccgcgagcca
     3001 ctgtggcgct tcccaaacga gctgccaatc gccggtgagc cagcgaacat cgtcgcgctg
     3061 gtcgaagaat acatggactg gctgcaccag tcccctgtcc cgaagctgct gttctggggc
     3121 accccaggcg ttctgatccc accggccgaa gccgctcgcc tggccaaaag cctgcctaac
     3181 tgcaaggctg tggacatcgg cccgggtctg aatctgctgc aagaagacaa cccggacctg
     3241 atcggcagcg agatcgcgcg ctggctgtct actctggaga tttccggtta atagaattct
     3301 agagtcgacc tgcaggcatg caagctgatt tggctgctgc caccgctgag caaggccgct
     3361 tcgagcagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     3421 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     3481 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     3541 gatgtgggag gtttttttaa gcaagtaaaa cctctacaaa tgtggtaaaa tcttaattaa
     3601 gaattctaat ataacttcgt atagcataca ttatacgaag ttatggatcc actgttgaca
     3661 attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaaccc
     3721 aggaggcaga tcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     3781 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     3841 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3901 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     3961 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4021 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4081 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4141 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4201 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4261 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4321 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     4381 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     4441 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     4501 tatcgccttc ttgacgagtt cttctgattc gaaatgaccg accaagcgac gcccaaccgg
     4561 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4621 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4681 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4741 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4801 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4861 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4921 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4981 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5041 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5101 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     5161 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5221 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggattt caagaagatc
     5281 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5341 tggtcatgag attatcaaaa aggatcttca cctagatcct tttatagtcc ggaatcgtca
     5401 cacaaaaaac caacacacag atgtaatgaa aataaagata ttttattgcg gccatcgtga
     5461 tggctagagt ttagccctcc cacacataac cagagggcag caattcacga atcccaactg
     5521 ccgtcggctg tccatcactg tccttcacta tggctttgat cccaggatgc agatcgagaa
     5581 gcacctgtcg gcaccgtccg caggggctca agatgcccct gttctcattt ccgatcgcga
     5641 cgatacaagt caggttgcca gctgccgcag cagcagcagt gcccagcacc acgagttctg
     5701 cacaaggtcc cccagtaaaa tgatatacat tgacaccagt gaagatgcgg ccgtcgctag
     5761 agagagctgc gctggcgacg ctgtagtctt cagagatggg gatgctgttg attgtagccg
     5821 ttgctctttc aatgagggtg gattcttctt gagacaaagg cttggccatg gtggcgctag
     5881 tgtcagaaga atcgagcttt ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact
     5941 tctggaatag ctcagaggcc gaggcggcct cggcctctgc ataaataaaa aaaattagtc
     6001 agccatgggg cggagaatgg gcggaactgg gcggagttag gggcgggatg ggcggagtta
     6061 ggggcgggac tatggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg
     6121 gggagcctgg ggactttcca cacctggttg ctgactaatt gagatgcatg ctttgcatac
     6181 ttctgcctgc tggggagcct ggggactttc cacaccctaa ctgacacaca ttccacagct
     6241 ggttctttcc gcctcagaag gtacctaacc aagttcctct ttcagaggtt atttcaggcc
     6301 atggtgctgc gcaagacgcg ttaattaaga cgt
//
