LOCUS       sequence-482032-        6969 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Express BTRC-HaloTag 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 6969)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6969)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: BTRC-HaloTag Vector
FEATURES             Location/Qualifiers
     source          1..6969
                     /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             3004..3024
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             3040..3924
                     /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    4011..4132
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4022..4041)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(4247..4280)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4290..4337
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4369..5163
                     /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(4423..4442)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5033..5052
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(5321..5909)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5401..5420)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    6035..6083
                     /note="synthetic polyadenylation signal"
     CDS             complement(6107..6505)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6502..6511
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6536..6893)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6550..6685
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6702..6893
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6850..6870
                     /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 gacccggccg aggcggtgct gcaagagaag
     1201 gcactcaagt ttatgtgctc tatgcccagg tctctgtggc tgggctgctc cagcctggcg
     1261 gacagcatgc cttcgctgcg atgcctgtat aacccaggga ctggcgcact cacagctttc
     1321 cagaattcct cagagagaga agactgtaat aatggcgaac cccctaggaa gataatacca
     1381 gagaagaatt cacttagaca gacatacaac agctgtgcca gactctgctt aaaccaagaa
     1441 acagtatgtt tagcaagcac tgctatgaag actgagaatt gtgtggccaa aacaaaactt
     1501 gccaatggca cttccagtat gattgtgccc aagcaacgga aactctcagc aagctatgaa
     1561 aaggaaaagg aactgtgtgt caaatacttt gagcagtggt cagagtcaga tcaagtggaa
     1621 tttgtggaac atcttatatc ccaaatgtgt cattaccaac atgggcacat aaactcgtat
     1681 cttaaaccta tgttgcagag agatttcata actgctctgc cagctcgggg attggatcat
     1741 attgctgaga acattctgtc atacctggat gccaaatcac tatgtgctgc tgaacttgtg
     1801 tgcaaggaat ggtaccgagt gacctctgat ggcatgctgt ggaagaagct tatcgagaga
     1861 atggtcagga cagattctct gtggagaggc ctggcagaac gaagaggatg gggacagtat
     1921 ttattcaaaa acaaacctcc tgacgggaat gctcctccca actcttttta tagagcactt
     1981 tatcctaaaa ttatacaaga cattgagaca atagaatcta attggagatg tggaagacat
     2041 agtttacaga gaattcactg ccgaagtgaa acaagcaaag gagtttactg tttacagtat
     2101 gatgatcaga aaatagtaag cggccttcga gacaacacaa tcaagatctg ggataaaaac
     2161 acattggaat gcaagcgaat tctcacaggc catacaggtt cagtcctctg tctccagtat
     2221 gatgagagag tgatcataac aggatcatcg gattccacgg tcagagtgtg ggatgtaaat
     2281 acaggtgaaa tgctaaacac gttgattcac cattgtgaag cagttctgca cttgcgtttc
     2341 aataatggca tgatggtgac ctgctccaaa gatcgttcca ttgctgtatg ggatatggcc
     2401 tccccaactg acattaccct ccggagggtg ctggtcggac accgagctgc tgtcaatgtt
     2461 gtagactttg atgacaagta cattgtttct gcatctgggg atagaactat aaaggtatgg
     2521 aacacaagta cttgtgaatt tgtaaggacc ttaaatggac acaaacgagg cattgcctgt
     2581 ttgcagtaca gggacaggct ggtagtgagt ggctcatctg acaacactat cagattatgg
     2641 gacatagaat gtggtgcatg tttacgagtg ttagaaggcc atgaggaatt ggtgcgttgt
     2701 attcgatttg ataacaagag gatagtcagt ggggcctatg atggaaaaat taaagtgtgg
     2761 gatcttgtgg ctgctttgga cccccgtgct cctgcaggga cactctgtct acggaccctt
     2821 gtggagcatt ccggaagagt ttttcgacta cagtttgatg aattccagat tgtcagtagt
     2881 tcacatgatg acacaatcct catctgggac ttcctaaatg atccagctgc ccaagctgaa
     2941 cccccccgtt ccccttctcg aacatacacc tacatctcca gagtttctct cgagccaacc
     3001 actgaggatc tgtactttca gagcgataac gatggatccg aaatcggtac tggctttcca
     3061 ttcgaccccc attatgtgga agtcctgggc gagcgcatgc actacgtcga tgttggtccg
     3121 cgcgatggca cccctgtgct gttcctgcac ggtaacccga cctcctccta cgtgtggcgc
     3181 aacatcatcc cgcatgttgc accgacccat cgctgcattg ctccagacct gatcggtatg
     3241 ggcaaatccg acaaaccaga cctgggttat ttcttcgacg accacgtccg cttcatggat
     3301 gccttcatcg aagccctggg tctggaagag gtcgtcctgg tcattcacga ctggggctcc
     3361 gctctgggtt tccactgggc caagcgcaat ccagagcgcg tcaaaggtat tgcatttatg
     3421 gagttcatcc gccctatccc gacctgggac gaatggccag aatttgcccg cgagaccttc
     3481 caggccttcc gcaccaccga cgtcggccgc aagctgatca tcgatcagaa cgtttttatc
     3541 gagggtacgc tgccgatggg tgtcgtccgc ccgctgactg aagtcgagat ggaccattac
     3601 cgcgagccgt tcctgaatcc tgttgaccgc gagccactgt ggcgcttccc aaacgagctg
     3661 ccaatcgccg gtgagccagc gaacatcgtc gcgctggtcg aagaatacat ggactggctg
     3721 caccagtccc ctgtcccgaa gctgctgttc tggggcaccc caggcgttct gatcccaccg
     3781 gccgaagccg ctcgcctggc caaaagcctg cctaactgca aggctgtgga catcggcccg
     3841 ggtctgaatc tgctgcaaga agacaacccg gacctgatcg gcagcgagat cgcgcgctgg
     3901 ctgtctactc tggagatttc cggttaatag aattctagag tcgacctgca ggcatgcaag
     3961 ctgatttggc tgctgccacc gctgagcaag gccgcttcga gcagacatga taagatacat
     4021 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     4081 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     4141 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt ttttaagcaa
     4201 gtaaaacctc tacaaatgtg gtaaaatctt aattaagaat tctaatataa cttcgtatag
     4261 catacattat acgaagttat ggatccactg ttgacaatta atcatcggca tagtatatcg
     4321 gcatagtata atacgacaag gtgaggaact aaacccagga ggcagatcat gattgaacaa
     4381 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     4441 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4501 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     4561 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     4621 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     4681 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     4741 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     4801 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     4861 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     4921 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     4981 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     5041 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     5101 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     5161 tgattcgaaa tgaccgacca agcgacgccc aaccggtatc agctcactca aaggcggtaa
     5221 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5281 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5341 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5401 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5461 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5521 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5581 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5641 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5701 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5761 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5821 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5881 agattacgcg cagaaaaaaa ggatttcaag aagatccttt gatcttttct acggggtctg
     5941 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6001 tcttcaccta gatcctttta tagtccggaa tcgtcacaca aaaaaccaac acacagatgt
     6061 aatgaaaata aagatatttt attgcggcca tcgtgatggc tagagtttag ccctcccaca
     6121 cataaccaga gggcagcaat tcacgaatcc caactgccgt cggctgtcca tcactgtcct
     6181 tcactatggc tttgatccca ggatgcagat cgagaagcac ctgtcggcac cgtccgcagg
     6241 ggctcaagat gcccctgttc tcatttccga tcgcgacgat acaagtcagg ttgccagctg
     6301 ccgcagcagc agcagtgccc agcaccacga gttctgcaca aggtccccca gtaaaatgat
     6361 atacattgac accagtgaag atgcggccgt cgctagagag agctgcgctg gcgacgctgt
     6421 agtcttcaga gatggggatg ctgttgattg tagccgttgc tctttcaatg agggtggatt
     6481 cttcttgaga caaaggcttg gccatggtgg cgctagtgtc agaagaatcg agctttttgc
     6541 aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg
     6601 cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg
     6661 aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact
     6721 aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc
     6781 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg
     6841 actttccaca ccctaactga cacacattcc acagctggtt ctttccgcct cagaaggtac
     6901 ctaaccaagt tcctctttca gaggttattt caggccatgg tgctgcgcaa gacgcgttaa
     6961 ttaagacgt
//
