LOCUS       sequence-470550-        5818 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express HaloTag(R)-VHL 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 5818)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5818)
  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            "Unpublished"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-VHL BlastR/KanR Vector
FEATURES             Location/Qualifiers
     source          1..5818
                     /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    2860..2981
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2871..2890)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3096..3129)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3139..3186
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3218..4012
                     /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(3272..3291)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3882..3901
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4170..4758)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4250..4269)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4884..4932
                     /note="synthetic polyadenylation signal"
     CDS             complement(4956..5354)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5351..5360
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5385..5742)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5399..5534
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5551..5742
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5699..5719
                     /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 gccccggagg gcggagaact gggacgaggc cgaggtaggc gcggaggagg
     2161 caggcgtcga agagtacggc cctgaagaag acggcgggga ggagtcgggc gccgaggagt
     2221 ccggcccgga agagtccggc ccggaggaac tgggcgccga ggaggagatg gaggccgggc
     2281 ggccgcggcc cgtgctgcgc tcggtgaact cgcgcgagcc ctcccaggtc atcttctgca
     2341 atcgcagtcc gcgcgtcgtg ctgcccgtat ggctcaactt cgacggcgag ccgcagccct
     2401 acccaacgct gccgcctggc acgggccgcc gcatccacag ctaccgaggt cacctttggc
     2461 tcttcagaga tgcagggaca cacgatgggc ttctggttaa ccaaactgaa ttatttgtgc
     2521 catctctcaa tgttgacgga cagcctattt ttgccaatat cacactgcca gtgtatactc
     2581 tgaaagagcg atgcctccag gttgtccgga gcctagtcaa gcctgagaat tacaggagac
     2641 tggacatcgt caggtcgctc tacgaagatc tggaagacca cccaaatgtg cagaaagacc
     2701 tggagcggct gacacaggag cgcattgcac atcaacggat gggagatgtt taaacgaatt
     2761 cgggctcggt acccggggat cctctagagt cgacctgcag gcatgcaagc tgatttggct
     2821 gctgccaccg ctgagcaagg ccgcttcgag cagacatgat aagatacatt gatgagtttg
     2881 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     2941 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     3001 attttatgtt tcaggttcag ggggagatgt gggaggtttt tttaagcaag taaaacctct
     3061 acaaatgtgg taaaatctta attaagaatt ctaatataac ttcgtatagc atacattata
     3121 cgaagttatg gatccactgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     3181 tacgacaagg tgaggaacta aacccaggag gcagatcatg attgaacaag atggattgca
     3241 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     3301 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     3361 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     3421 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     3481 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     3541 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     3601 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     3661 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     3721 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     3781 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     3841 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     3901 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     3961 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gattcgaaat
     4021 gaccgaccaa gcgacgccca accggtatca gctcactcaa aggcggtaat acggttatcc
     4081 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4141 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4201 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4261 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4321 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4381 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4441 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4501 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4561 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     4621 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4681 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4741 agaaaaaaag gatttcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4801 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4861 atccttttat agtccggaat cgtcacacaa aaaaccaaca cacagatgta atgaaaataa
     4921 agatatttta ttgcggccat cgtgatggct agagtttagc cctcccacac ataaccagag
     4981 ggcagcaatt cacgaatccc aactgccgtc ggctgtccat cactgtcctt cactatggct
     5041 ttgatcccag gatgcagatc gagaagcacc tgtcggcacc gtccgcaggg gctcaagatg
     5101 cccctgttct catttccgat cgcgacgata caagtcaggt tgccagctgc cgcagcagca
     5161 gcagtgccca gcaccacgag ttctgcacaa ggtcccccag taaaatgata tacattgaca
     5221 ccagtgaaga tgcggccgtc gctagagaga gctgcgctgg cgacgctgta gtcttcagag
     5281 atggggatgc tgttgattgt agccgttgct ctttcaatga gggtggattc ttcttgagac
     5341 aaaggcttgg ccatggtggc gctagtgtca gaagaatcga gctttttgca aaagcctagg
     5401 cctccaaaaa agcctcctca ctacttctgg aatagctcag aggccgaggc ggcctcggcc
     5461 tctgcataaa taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga
     5521 gttaggggcg ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc
     5581 atgctttgca tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac
     5641 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     5701 cctaactgac acacattcca cagctggttc tttccgcctc agaaggtacc taaccaagtt
     5761 cctctttcag aggttatttc aggccatggt gctgcgcaag acgcgttaat taagacgt
//
