LOCUS       sequence-472397-        6742 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express HaloTag(R)-PPP3CA 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 6742)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6742)
  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            "Unpublished"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: HaloTag-PPP3CA Vector
FEATURES             Location/Qualifiers
     source          1..6742
                     /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"
     polyA_signal    3784..3905
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3795..3814)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(4020..4053)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4063..4110
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4142..4936
                     /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(4196..4215)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4806..4825
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(5094..5682)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5174..5193)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5808..5856
                     /note="synthetic polyadenylation signal"
     CDS             complement(5880..6278)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6275..6284
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6309..6666)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6323..6458
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6475..6666
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6623..6643
                     /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 gtccgagccc aaggcaattg atcccaagtt gtcgacgacc gacagggtgg
     2161 tgaaagctgt tccatttcct ccaagtcacc ggcttacagc aaaagaagtg tttgataatg
     2221 atggaaaacc tcgtgtggat atcttaaagg cgcatcttat gaaggaggga aggctggaag
     2281 agagtgttgc attgagaata ataacagagg gtgcatcaat tcttcgacag gaaaaaaatt
     2341 tgctggatat tgatgcgcca gtcactgttt gtggggacat tcatggacaa ttctttgatt
     2401 tgatgaagct ctttgaagtc gggggatctc ctgccaacac tcgctacctc ttcttagggg
     2461 actatgttga cagagggtac ttcagtattg aatgtgtgct gtatttgtgg gccttgaaaa
     2521 ttctctaccc caaaacactg tttttacttc gtggaaatca tgaatgtaga catctaacag
     2581 agtatttcac atttaaacaa gaatgtaaaa taaagtattc agaacgcgta tatgatgcct
     2641 gtatggatgc ctttgactgc cttcccctgg ctgccctgat gaaccaacag ttcctgtgtg
     2701 tgcatggtgg tttgtctcca gagattaaca ctttagatga tatcagaaaa ttagaccgat
     2761 tcaaagaacc acctgcatat ggacctatgt gtgatatcct gtggtcagac cccctggaag
     2821 attttggaaa tgagaagact caggaacatt tcactcacaa cacagtcagg gggtgttcat
     2881 acttctacag ttacccggct gtatgtgaat tcttacagca caataacttg ttatctatac
     2941 tccgagccca cgaagcccaa gatgcagggt accgcatgta caggaaaagc caaacaacag
     3001 gcttcccttc tctaattaca attttttcag caccaaatta cttagatgta tacaataaca
     3061 aagctgcagt attgaagtat gagaacaatg ttatgaatat caggcaattc aactgttctc
     3121 ctcatccata ctggcttcca aatttcatgg atgtttttac ttggtccctt ccatttgttg
     3181 gggaaaaagt gactgagatg ctggtaaatg tcctcaacat ctgctcagat gatgaactag
     3241 ggtcagaaga agatggattt gatggtgcaa cagctgcagc ccggaaagag gtgataagga
     3301 acaagatccg agcaataggc aaaatggcca gagtgttctc agtgctcaga gaagagagtg
     3361 agagtgtgct gacgctgaaa ggcttgaccc caactggcat gctccccagc ggagtacttt
     3421 ctggagggaa gcaaaccctg caaagcgcta ctgttgaggc tattgaggct gatgaagcta
     3481 tcaaaggatt ttcaccacaa cataagatca ctagcttcga ggaagccaag ggcttagacc
     3541 gaattaatga gaggatgccg cctcgcagag atgccatgcc ctctgacgcc aaccttaact
     3601 ccatcaacaa ggctctcacc tcagagacta acggcacgga cagcaatggc agtaatagca
     3661 gcaatattca ggtttaaacg aattcgggct cggtacccgg ggatcctcta gagtcgacct
     3721 gcaggcatgc aagctgattt ggctgctgcc accgctgagc aaggccgctt cgagcagaca
     3781 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     3841 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     3901 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg
     3961 tttttttaag caagtaaaac ctctacaaat gtggtaaaat cttaattaag aattctaata
     4021 taacttcgta tagcatacat tatacgaagt tatggatcca ctgttgacaa ttaatcatcg
     4081 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccca ggaggcagat
     4141 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4201 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4261 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4321 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4381 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4441 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4501 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4561 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4621 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4681 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4741 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4801 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4861 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4921 tgacgagttc ttctgattcg aaatgaccga ccaagcgacg cccaaccggt atcagctcac
     4981 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5041 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5101 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5161 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5221 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5281 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5341 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5401 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5461 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5521 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5581 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5641 gtttgcaagc agcagattac gcgcagaaaa aaaggatttc aagaagatcc tttgatcttt
     5701 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5761 ttatcaaaaa ggatcttcac ctagatcctt ttatagtccg gaatcgtcac acaaaaaacc
     5821 aacacacaga tgtaatgaaa ataaagatat tttattgcgg ccatcgtgat ggctagagtt
     5881 tagccctccc acacataacc agagggcagc aattcacgaa tcccaactgc cgtcggctgt
     5941 ccatcactgt ccttcactat ggctttgatc ccaggatgca gatcgagaag cacctgtcgg
     6001 caccgtccgc aggggctcaa gatgcccctg ttctcatttc cgatcgcgac gatacaagtc
     6061 aggttgccag ctgccgcagc agcagcagtg cccagcacca cgagttctgc acaaggtccc
     6121 ccagtaaaat gatatacatt gacaccagtg aagatgcggc cgtcgctaga gagagctgcg
     6181 ctggcgacgc tgtagtcttc agagatgggg atgctgttga ttgtagccgt tgctctttca
     6241 atgagggtgg attcttcttg agacaaaggc ttggccatgg tggcgctagt gtcagaagaa
     6301 tcgagctttt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc
     6361 tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc
     6421 ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact
     6481 atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     6541 gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct
     6601 ggggagcctg gggactttcc acaccctaac tgacacacat tccacagctg gttctttccg
     6661 cctcagaagg tacctaacca agttcctctt tcagaggtta tttcaggcca tggtgctgcg
     6721 caagacgcgt taattaagac gt
//
