LOCUS       sequence-475170-        6612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express PPP2R5A-HaloTag 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 6612)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6612)
  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            "Unpublished"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: NanoBRET Assay Vector, PPP2R5A-HaloTag
FEATURES             Location/Qualifiers
     source          1..6612
                     /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             2647..2667
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             2683..3567
                     /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    3654..3775
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3665..3684)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3890..3923)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3933..3980
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4012..4806
                     /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(4066..4085)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4676..4695
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4964..5552)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5044..5063)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5678..5726
                     /note="synthetic polyadenylation signal"
     CDS             complement(5750..6148)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6145..6154
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6179..6536)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6193..6328
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6345..6536
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6493..6513
                     /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 tcgtcgtcgt cgccgccggc gggggctgcc
     1201 agcgccgcca tctcggcctc ggagaaagtg gacggcttca cccggaaatc ggtccgcaag
     1261 gcgcagaggc agaagcgctc ccagggctcg tcgcagtttc gcagccaggg cagccaggca
     1321 gagctgcacc cgctgcccca gctcaaagat gccacttcaa atgaacaaca agagcttttc
     1381 tgtcagaagt tgcagcagtg ttgtatactg tttgatttca tggactctgt ttcagacttg
     1441 aagagcaaag aaattaaaag agcaacactg aatgaactgg ttgagtatgt ttcaactaat
     1501 cgtggtgtaa ttgttgaatc agcgtattct gatatagtaa aaatgatcag tgctaacatc
     1561 ttccgtacac ttcctccaag tgataatcca gattttgatc cagaagagga tgaacccacg
     1621 cttgaggcct cttggcctca catacagttg gtatatgaat tcttcttgag atttttggag
     1681 agccctgatt tccagcctag cattgcaaaa cgatacattg atcagaaatt cgtacaacag
     1741 ctcctggagc tttttgatag tgaagatccc agagaacgtg acttcctgaa gactgttctg
     1801 caccgaattt atgggaaatt tcttggatta agagcattca tcagaaaaca aattaacaac
     1861 attttcctca ggtttatata tgaaacagaa catttcaatg gtgttgctga acttcttgaa
     1921 atattaggaa gtattatcaa tggctttgca ttgccactga aagcagaaca taaacaattt
     1981 ctaatgaagg ttcttattcc tatgcatact gcaaaaggat tagctttgtt tcatgctcag
     2041 ctagcatatt gtgttgtaca gttcctggag aaagatacaa cactaacaga gccagtgatc
     2101 agaggactgc tgaaattttg gccaaaaacc tgcagtcaga aagaggtgat gtttttagga
     2161 gaaattgaag aaatcttaga tgtcattgaa ccaacacagt tcaaaaaaat tgaagagcca
     2221 cttttcaagc agatatccaa gtgtgtatcc agttctcatt ttcaggttgc agaaagggca
     2281 ttgtacttct ggaataacga atatattctt agtttgattg aggagaacat tgataaaatt
     2341 ctgccaatta tgtttgccag tttgtacaaa atttccaaag aacactggaa tccgaccatt
     2401 gtagcactgg tatacaatgt gctgaaaacc ctaatggaaa tgaatggcaa gcttttcgat
     2461 gaccttacta gctcatacaa agctgaaaga cagagagaga aaaagaagga attggaacgt
     2521 gaagaattat ggaaaaaatt agaggagcta aagctaaaga aagctctaga aaaacagaat
     2581 agtgcttaca acatgcacag tattctcagc aatacaagtg ccgaagtttc tctcgagcca
     2641 accactgagg atctgtactt tcagagcgat aacgatggat ccgaaatcgg tactggcttt
     2701 ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt
     2761 ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg
     2821 cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga cctgatcggt
     2881 atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt ccgcttcatg
     2941 gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca cgactggggc
     3001 tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg tattgcattt
     3061 atggagttca tccgccctat cccgacctgg gacgaatggc cagaatttgc ccgcgagacc
     3121 ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca gaacgttttt
     3181 atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat
     3241 taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt cccaaacgag
     3301 ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata catggactgg
     3361 ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt tctgatccca
     3421 ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc
     3481 ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga gatcgcgcgc
     3541 tggctgtcta ctctggagat ttccggttaa tagaattcta gagtcgacct gcaggcatgc
     3601 aagctgattt ggctgctgcc accgctgagc aaggccgctt cgagcagaca tgataagata
     3661 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     3721 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     3781 caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg tttttttaag
     3841 caagtaaaac ctctacaaat gtggtaaaat cttaattaag aattctaata taacttcgta
     3901 tagcatacat tatacgaagt tatggatcca ctgttgacaa ttaatcatcg gcatagtata
     3961 tcggcatagt ataatacgac aaggtgagga actaaaccca ggaggcagat catgattgaa
     4021 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4081 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4141 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     4201 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4261 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4321 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4381 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4441 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4501 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     4561 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4621 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4681 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4741 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4801 ttctgattcg aaatgaccga ccaagcgacg cccaaccggt atcagctcac tcaaaggcgg
     4861 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4921 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4981 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5041 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5101 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5161 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5221 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5281 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5341 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5401 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5461 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5521 agcagattac gcgcagaaaa aaaggatttc aagaagatcc tttgatcttt tctacggggt
     5581 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5641 ggatcttcac ctagatcctt ttatagtccg gaatcgtcac acaaaaaacc aacacacaga
     5701 tgtaatgaaa ataaagatat tttattgcgg ccatcgtgat ggctagagtt tagccctccc
     5761 acacataacc agagggcagc aattcacgaa tcccaactgc cgtcggctgt ccatcactgt
     5821 ccttcactat ggctttgatc ccaggatgca gatcgagaag cacctgtcgg caccgtccgc
     5881 aggggctcaa gatgcccctg ttctcatttc cgatcgcgac gatacaagtc aggttgccag
     5941 ctgccgcagc agcagcagtg cccagcacca cgagttctgc acaaggtccc ccagtaaaat
     6001 gatatacatt gacaccagtg aagatgcggc cgtcgctaga gagagctgcg ctggcgacgc
     6061 tgtagtcttc agagatgggg atgctgttga ttgtagccgt tgctctttca atgagggtgg
     6121 attcttcttg agacaaaggc ttggccatgg tggcgctagt gtcagaagaa tcgagctttt
     6181 tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggccg
     6241 aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg
     6301 cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg
     6361 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
     6421 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     6481 gggactttcc acaccctaac tgacacacat tccacagctg gttctttccg cctcagaagg
     6541 tacctaacca agttcctctt tcagaggtta tttcaggcca tggtgctgcg caagacgcgt
     6601 taattaagac gt
//
