LOCUS       sequence-479894-        6946 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express HaloTag-PPP2R1A 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 6946)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6946)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 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            "Unpublished"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, HaloTag-PPP2R1A
FEATURES             Location/Qualifiers
     source          1..6946
                     /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"
     regulatory      1164..1173
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1170..2060
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             2073..2093
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     regulatory      2107..2116
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3988..4109
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3999..4018)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(4224..4257)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4267..4314
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4346..5140
                     /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(4400..4419)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5010..5029
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(5298..5886)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5378..5397)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    6012..6060
                     /note="synthetic polyadenylation signal"
     CDS             complement(6084..6482)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6479..6488
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6513..6870)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6527..6662
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6679..6870
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6827..6847
                     /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 aaagccacca tggcagaaat cggtactggc tttccattcg
     1201 acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg
     1261 atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca
     1321 tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca
     1381 aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct
     1441 tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc
     1501 tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt
     1561 tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag accttccagg
     1621 ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg
     1681 gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg
     1741 agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa
     1801 tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc
     1861 agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg
     1921 aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc
     1981 tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt
     2041 cgacgctcga gatttccggc gagccaacca ctgaggatct gtactttcag agcgataacg
     2101 cgatcgccat ggcggcggcc gacggcgacg actcgctgta ccccatcgcg gtgctcatag
     2161 acgaactccg caatgaggac gttcagcttc gcctcaacag catcaagaag ctgtccacca
     2221 tcgccttggc ccttggggtt gaaaggaccc gaagtgagct tctgcctttc cttacagata
     2281 ccatctatga tgaagatgag gtcctcctgg ccctggcaga acagctggga accttcacta
     2341 ccctggtggg aggcccagag tacgtgcact gcctgctgcc accgctggag tcgctggcca
     2401 cagtggagga gacagtggtg cgggacaagg cagtggagtc cttacgggcc atctcacacg
     2461 agcactcgcc ctctgacctg gaggcgcact ttgtgccgct agtgaagcgg ctggcgggcg
     2521 gcgactggtt cacctcccgc acctcggcct gcggcctctt ctccgtctgc tacccccgag
     2581 tgtccagtgc tgtgaaggcg gaacttcgac agtacttccg gaacctgtgc tcagatgaca
     2641 cccccatggt gcggcgggcc gcagcctcca agctggggga gtttgccaag gtgctggagc
     2701 tggacaacgt caagagtgag atcatcccca tgttctccaa cctggcctct gacgagcagg
     2761 actcggtgcg gctgctggcg gtggaggcgt gcgtgaacat cgcccagctt ctgccccagg
     2821 aggatctgga ggccctggtg atgcccactc tgcgccaggc cgctgaagac aagtcctggc
     2881 gcgtccgcta catggtggct gacaagttca cagagctcca gaaagcagtg gggcctgaga
     2941 tcaccaagac agacctggtc cctgccttcc agaacctgat gaaagactgt gaggccgagg
     3001 tgagggccgc agcctcccac aaggtcaaag agttctgtga aaacctctca gctgactgtc
     3061 gggagaatgt gatcatgtcc cagatcttgc cctgcatcaa ggagctggtg tccgatgcca
     3121 accaacatgt caagtctgcc ctggcctcag tcatcatggg tctctctccc atcttgggca
     3181 aagacaacac catcgagcac ctcttgcccc tcttcctggc tcagctgaag gatgagtgcc
     3241 ctgaggtacg gctgaacatc atctctaacc tggactgtgt gaacgaggtg attggcatcc
     3301 ggcagctgtc ccagtccctg ctccctgcca ttgtggagct ggctgaggac gccaagtggc
     3361 gggtgcggct ggccatcatt gagtacatgc ccctcctggc tggacagctg ggagtggagt
     3421 tctttgatga gaaacttaac tccttgtgca tggcctggct tgtggatcat gtatatgcca
     3481 tccgcgaggc agccaccagc aacctgaaga agctagtgga aaagtttggg aaggagtggg
     3541 cccatgccac aatcatcccc aaggtcttgg ccatgtccgg agaccccaac tacctgcacc
     3601 gcatgactac gctcttctgc atcaatgtgc tgtctgaggt ctgtgggcag gacatcacca
     3661 ccaagcacat gctacccacg gttctgcgca tggctgggga cccggttgcc aatgtccgct
     3721 tcaatgtggc caagtctctg cagaagatag ggcccatcct ggacaacagc accttgcaga
     3781 gtgaagtcaa gcccatccta gagaagctga cccaggacca ggatgtggac gtcaaatact
     3841 ttgcccagga ggctctgact gttctgtctc tcgccgttta aacgaattcg ggctcggtac
     3901 ccggggatcc tctagagtcg acctgcaggc atgcaagctg atttggctgc tgccaccgct
     3961 gagcaaggcc gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa
     4021 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     4081 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     4141 aggttcaggg ggagatgtgg gaggtttttt taagcaagta aaacctctac aaatgtggta
     4201 aaatcttaat taagaattct aatataactt cgtatagcat acattatacg aagttatgga
     4261 tccactgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg
     4321 aggaactaaa cccaggaggc agatcatgat tgaacaagat ggattgcacg caggttctcc
     4381 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4441 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4501 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     4561 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4621 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4681 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4741 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4801 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4861 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4921 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4981 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     5041 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     5101 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga ttcgaaatga ccgaccaagc
     5161 gacgcccaac cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5221 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5281 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5341 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5401 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5461 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5521 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5581 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5641 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5701 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     5761 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5821 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5881 tttcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5941 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttatag
     6001 tccggaatcg tcacacaaaa aaccaacaca cagatgtaat gaaaataaag atattttatt
     6061 gcggccatcg tgatggctag agtttagccc tcccacacat aaccagaggg cagcaattca
     6121 cgaatcccaa ctgccgtcgg ctgtccatca ctgtccttca ctatggcttt gatcccagga
     6181 tgcagatcga gaagcacctg tcggcaccgt ccgcaggggc tcaagatgcc cctgttctca
     6241 tttccgatcg cgacgataca agtcaggttg ccagctgccg cagcagcagc agtgcccagc
     6301 accacgagtt ctgcacaagg tcccccagta aaatgatata cattgacacc agtgaagatg
     6361 cggccgtcgc tagagagagc tgcgctggcg acgctgtagt cttcagagat ggggatgctg
     6421 ttgattgtag ccgttgctct ttcaatgagg gtggattctt cttgagacaa aggcttggcc
     6481 atggtggcgc tagtgtcaga agaatcgagc tttttgcaaa agcctaggcc tccaaaaaag
     6541 cctcctcact acttctggaa tagctcagag gccgaggcgg cctcggcctc tgcataaata
     6601 aaaaaaatta gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg
     6661 atgggcggag ttaggggcgg gactatggtt gctgactaat tgagatgcat gctttgcata
     6721 cttctgcctg ctggggagcc tggggacttt ccacacctgg ttgctgacta attgagatgc
     6781 atgctttgca tacttctgcc tgctggggag cctggggact ttccacaccc taactgacac
     6841 acattccaca gctggttctt tccgcctcag aaggtaccta accaagttcc tctttcagag
     6901 gttatttcag gccatggtgc tgcgcaagac gcgttaatta agacgt
//
