LOCUS       sequence-473351-        6465 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  Express hVPS4A-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 6465)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 6465)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: hVPS4A-HaloTag Vector
FEATURES             Location/Qualifiers
     source          1..6465
                     /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             2500..2520
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             2536..3420
                     /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    3507..3628
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3518..3537)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3743..3776)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3786..3833
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3865..4659
                     /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(3919..3938)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4529..4548
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4817..5405)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4897..4916)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5531..5579
                     /note="synthetic polyadenylation signal"
     CDS             complement(5603..6001)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5998..6007
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6032..6389)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6046..6181
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6198..6389
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6346..6366
                     /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 acaacgtcaa ccctccagaa agccattgat
     1201 ctggtgacga aagccacaga ggaggacaaa gccaagaact acgaggaggc gctgcggctg
     1261 taccagcatg cggtggagta cttcctccac gctatcaagt atgaggccca cagcgacaag
     1321 gccaaggaga gcattcgagc caagtgcgtg cagtacctag accgggccga gaagctgaag
     1381 gattatttac gaagcaaaga gaaacacggc aagaagccag tcaaagagaa ccagagtgag
     1441 ggcaagggca gtgacagtga cagtgaaggg gataatccgg agaaaaagaa actgcaagaa
     1501 cagctgatgg gtgccgtcgt gatggagaag cccaacatac ggtggaacga cgtggccggg
     1561 ctggaggggg ccaaggaggc cctcaaagaa gctgtcattt tgccaatcaa attcccacac
     1621 ttgttcacag gcaagcgcac cccctggcgg gggattctgc tgttcggacc ccctggcaca
     1681 gggaaatcct acctggccaa agccgtggca acagaggcca acaactccac cttcttctct
     1741 gtgtcctcct cagatctgat gtccaagtgg ctgggggaga gtgaaaagct ggtcaagaac
     1801 ctgtttgagc tggccaggca gcacaagccc tccatcatct tcatcgatga ggtggattcc
     1861 ctctgcgggt cccgaaatga aaatgagagt gaggccgccc ggaggatcaa aacggagttc
     1921 ttggtccaga tgcagggggt ggggaataac aatgatggga ctctggttct tggagccaca
     1981 aacatcccat gggtgttgga ttcggccatc aggaggaggt ttgaaaaacg aatttatatc
     2041 cccttgccgg aggaagctgc ccgcgcccag atgttccggt tgcatctcgg gagcactccc
     2101 cacaacctca cggatgcaaa catccacgag ctggcccgga agacggaagg ctactcgggc
     2161 gcggacatca gcatcatcgt gcgggactct ctcatgcagc ccgtgaggaa ggtgcagtcg
     2221 gccacacact tcaaaaaggt ctgtggcccc tctcgcacca accccagcat gatgattgat
     2281 gacctcctga ctccatgctc accaggggac ccaggagcca tggagatgac ttggatggat
     2341 gtccctgggg acaaactctt agagcctgtg gtttgcatgt cggacatgct gcggtctctg
     2401 gccaccaccc ggcccacggt gaatgcagac gacctcctga aagtgaagaa attctcagag
     2461 gactttgggc aagagagtgt ttctctcgag ccaaccactg aggatctgta ctttcagagc
     2521 gataacgatg gatccgaaat cggtactggc tttccattcg acccccatta tgtggaagtc
     2581 ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc
     2641 ctgcacggta acccgacctc ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg
     2701 acccatcgct gcattgctcc agacctgatc ggtatgggca aatccgacaa accagacctg
     2761 ggttatttct tcgacgacca cgtccgcttc atggatgcct tcatcgaagc cctgggtctg
     2821 gaagaggtcg tcctggtcat tcacgactgg ggctccgctc tgggtttcca ctgggccaag
     2881 cgcaatccag agcgcgtcaa aggtattgca tttatggagt tcatccgccc tatcccgacc
     2941 tgggacgaat ggccagaatt tgcccgcgag accttccagg ccttccgcac caccgacgtc
     3001 ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc gatgggtgtc
     3061 gtccgcccgc tgactgaagt cgagatggac cattaccgcg agccgttcct gaatcctgtt
     3121 gaccgcgagc cactgtggcg cttcccaaac gagctgccaa tcgccggtga gccagcgaac
     3181 atcgtcgcgc tggtcgaaga atacatggac tggctgcacc agtcccctgt cccgaagctg
     3241 ctgttctggg gcaccccagg cgttctgatc ccaccggccg aagccgctcg cctggccaaa
     3301 agcctgccta actgcaaggc tgtggacatc ggcccgggtc tgaatctgct gcaagaagac
     3361 aacccggacc tgatcggcag cgagatcgcg cgctggctgt ctactctgga gatttccggt
     3421 taatagaatt ctagagtcga cctgcaggca tgcaagctga tttggctgct gccaccgctg
     3481 agcaaggccg cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac
     3541 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     3601 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     3661 ggttcagggg gagatgtggg aggttttttt aagcaagtaa aacctctaca aatgtggtaa
     3721 aatcttaatt aagaattcta atataacttc gtatagcata cattatacga agttatggat
     3781 ccactgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga
     3841 ggaactaaac ccaggaggca gatcatgatt gaacaagatg gattgcacgc aggttctccg
     3901 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3961 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4021 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     4081 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4141 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4201 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4261 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4321 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4381 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4441 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4501 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4561 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4621 cgcatcgcct tctatcgcct tcttgacgag ttcttctgat tcgaaatgac cgaccaagcg
     4681 acgcccaacc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4741 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4801 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4861 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4921 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4981 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5041 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5101 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5161 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5221 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     5281 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5341 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5401 ttcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5461 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttatagt
     5521 ccggaatcgt cacacaaaaa accaacacac agatgtaatg aaaataaaga tattttattg
     5581 cggccatcgt gatggctaga gtttagccct cccacacata accagagggc agcaattcac
     5641 gaatcccaac tgccgtcggc tgtccatcac tgtccttcac tatggctttg atcccaggat
     5701 gcagatcgag aagcacctgt cggcaccgtc cgcaggggct caagatgccc ctgttctcat
     5761 ttccgatcgc gacgatacaa gtcaggttgc cagctgccgc agcagcagca gtgcccagca
     5821 ccacgagttc tgcacaaggt cccccagtaa aatgatatac attgacacca gtgaagatgc
     5881 ggccgtcgct agagagagct gcgctggcga cgctgtagtc ttcagagatg gggatgctgt
     5941 tgattgtagc cgttgctctt tcaatgaggg tggattcttc ttgagacaaa ggcttggcca
     6001 tggtggcgct agtgtcagaa gaatcgagct ttttgcaaaa gcctaggcct ccaaaaaagc
     6061 ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa
     6121 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
     6181 tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac
     6241 ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca
     6301 tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca
     6361 cattccacag ctggttcttt ccgcctcaga aggtacctaa ccaagttcct ctttcagagg
     6421 ttatttcagg ccatggtgct gcgcaagacg cgttaattaa gacgt
//
