LOCUS       sequence-473318-        6075 bp    DNA     circular SYN 28-MAY-2025
DEFINITION  Express hVTA1-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 6075)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 6075)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 28, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: hVTA1-HaloTag Vector
FEATURES             Location/Qualifiers
     source          1..6075
                     /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             2110..2130
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             2146..3030
                     /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    3117..3238
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3128..3147)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3353..3386)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3396..3443
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3475..4269
                     /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(3529..3548)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4139..4158
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4427..5015)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4507..4526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5141..5189
                     /note="synthetic polyadenylation signal"
     CDS             complement(5213..5611)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5608..5617
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5642..5999)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5656..5791
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5808..5999
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5956..5976
                     /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 gccgcgcttg caccgctgcc cccgctcccc
     1201 gcacagttca agagcataca gcatcatctg aggacggctc aggagcatga caagcgagac
     1261 cctgtggtgg cttattactg tcgtttatac gcaatgcaga ctggaatgaa gatcgatagt
     1321 aaaactcctg aatgtcgcaa atttttatca aagttaatgg atcagttaga agctctaaag
     1381 aagcagttgg gtgataatga agctattact caagaaatag tgggctgtgc ccatttggag
     1441 aattatgctt tgaaaatgtt tttgtatgca gacaatgaag atcgtgctgg acgatttcac
     1501 aaaaacatga tcaagtcctt ctatactgca agtcttttga tagatgtcat aacagtattt
     1561 ggagaactca ctgatgaaaa tgtgaaacac aggaagtatg ccagatggaa ggcaacatac
     1621 atccataatt gtttaaagaa tggggagact cctcaagcag gccctgttgg aattgaagaa
     1681 gataatgata ttgaagaaaa tgaagatgct ggagcagcct ctctgcccac tcagccaact
     1741 cagccatcat catcttcaac ttatgaccca agcaacatgc catcaggcaa ctatactgga
     1801 atacagattc ctccgggtgc acacgctcca gctaatacac cagcagaagt gcctcacagc
     1861 acaggtgtag caagtaatac tatccaacct actccacaga ctatacctgc cattgatccc
     1921 gcacttttca atacaatttc ccagggggat gttcgtctaa ccccagaaga ctttgctaga
     1981 gctcagaagt actgcaaata tgctggcagt gctttgcagt atgaagatgt aagcactgct
     2041 gtccagaatc tacaaaaggc tctcaagtta ctgacgacag gcagagaagt ttctctcgag
     2101 ccaaccactg aggatctgta ctttcagagc gataacgatg gatccgaaat cggtactggc
     2161 tttccattcg acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt
     2221 ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg
     2281 tggcgcaaca tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc
     2341 ggtatgggca aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc
     2401 atggatgcct tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg
     2461 ggctccgctc tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca
     2521 tttatggagt tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag
     2581 accttccagg ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt
     2641 tttatcgagg gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac
     2701 cattaccgcg agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac
     2761 gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac
     2821 tggctgcacc agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc
     2881 ccaccggccg aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc
     2941 ggcccgggtc tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg
     3001 cgctggctgt ctactctgga gatttccggt taatagaatt ctagagtcga cctgcaggca
     3061 tgcaagctga tttggctgct gccaccgctg agcaaggccg cttcgagcag acatgataag
     3121 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     3181 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     3241 caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggttttttt
     3301 aagcaagtaa aacctctaca aatgtggtaa aatcttaatt aagaattcta atataacttc
     3361 gtatagcata cattatacga agttatggat ccactgttga caattaatca tcggcatagt
     3421 atatcggcat agtataatac gacaaggtga ggaactaaac ccaggaggca gatcatgatt
     3481 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     3541 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     3601 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     3661 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     3721 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     3781 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     3841 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     3901 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     3961 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     4021 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     4081 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4141 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4201 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4261 ttcttctgat tcgaaatgac cgaccaagcg acgcccaacc ggtatcagct cactcaaagg
     4321 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4381 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4441 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4501 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4561 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4621 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4681 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4741 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4801 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4861 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4921 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4981 agcagcagat tacgcgcaga aaaaaaggat ttcaagaaga tcctttgatc ttttctacgg
     5041 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5101 aaaggatctt cacctagatc cttttatagt ccggaatcgt cacacaaaaa accaacacac
     5161 agatgtaatg aaaataaaga tattttattg cggccatcgt gatggctaga gtttagccct
     5221 cccacacata accagagggc agcaattcac gaatcccaac tgccgtcggc tgtccatcac
     5281 tgtccttcac tatggctttg atcccaggat gcagatcgag aagcacctgt cggcaccgtc
     5341 cgcaggggct caagatgccc ctgttctcat ttccgatcgc gacgatacaa gtcaggttgc
     5401 cagctgccgc agcagcagca gtgcccagca ccacgagttc tgcacaaggt cccccagtaa
     5461 aatgatatac attgacacca gtgaagatgc ggccgtcgct agagagagct gcgctggcga
     5521 cgctgtagtc ttcagagatg gggatgctgt tgattgtagc cgttgctctt tcaatgaggg
     5581 tggattcttc ttgagacaaa ggcttggcca tggtggcgct agtgtcagaa gaatcgagct
     5641 ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg
     5701 ccgaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat
     5761 gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg actatggttg
     5821 ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc
     5881 cacacctggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc
     5941 ctggggactt tccacaccct aactgacaca cattccacag ctggttcttt ccgcctcaga
     6001 aggtacctaa ccaagttcct ctttcagagg ttatttcagg ccatggtgct gcgcaagacg
     6061 cgttaattaa gacgt
//
