LOCUS       sequence-482040-        6447 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Express BTRC(del)-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 6447)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6447)
  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            "Unpublished"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(del)-HaloTag Vector
FEATURES             Location/Qualifiers
     source          1..6447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        144..501
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        144..335
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(167..187)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      352..487
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      526..535
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             532..930
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    954..1002
                     /note="synthetic polyadenylation signal"
     rep_origin      1128..1716
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1617..1636
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1874..2668)
                     /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(1985..2004)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2595..2614
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2700..2747)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     protein_bind    2757..2790
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2905..3026
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     2996..3015
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(3113..3997)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             complement(4013..4033)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(5360..5379)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5361..5379)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5421..5440)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          5423..5555
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        complement(5691..5894)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5720..5740)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5895..6198
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    join(6431..6447,1..65)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
       61 actgccgacg tcttaattaa cgcgtcttgc gcagcaccat ggcctgaaat aacctctgaa
      121 agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
      181 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
      241 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
      301 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
      361 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
      421 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
      481 ggaggcctag gcttttgcaa aaagctcgat tcttctgaca ctagcgccac catggccaag
      541 cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat caacagcatc
      601 cccatctctg aagactacag cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc
      661 actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt ggtgctgggc
      721 actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac
      781 aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct gcatcctggg
      841 atcaaagcca tagtgaagga cagtgatgga cagccgacgg cagttgggat tcgtgaattg
      901 ctgccctctg gttatgtgtg ggagggctaa actctagcca tcacgatggc cgcaataaaa
      961 tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgacgattcc ggactataaa
     1021 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     1081 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg aaatcctttt
     1141 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     1201 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     1261 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     1321 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     1381 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     1441 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     1501 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     1561 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     1621 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     1681 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     1741 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     1801 tctgtggata accgtattac cgcctttgag tgagctgata ccggttgggc gtcgcttggt
     1861 cggtcatttc gaatcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat
     1921 cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt
     1981 cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc
     2041 cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat
     2101 cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca
     2161 gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg
     2221 cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg
     2281 tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg
     2341 caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt
     2401 cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca
     2461 gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct
     2521 tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc
     2581 cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac
     2641 ctgcgtgcaa tccatcttgt tcaatcatga tctgcctcct gggtttagtt cctcaccttg
     2701 tcgtattata ctatgccgat atactatgcc gatgattaat tgtcaacagt ggatccataa
     2761 cttcgtataa tgtatgctat acgaagttat attagaattc ttaattaaga ttttaccaca
     2821 tttgtagagg ttttacttgc ttaaaaaaac ctcccacatc tccccctgaa cctgaaacat
     2881 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     2941 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3001 ttgtccaaac tcatcaatgt atcttatcat gtctgctcga agcggccttg ctcagcggtg
     3061 gcagcagcca aatcagcttg catgcctgca ggtcgactct agaattctat taaccggaaa
     3121 tctccagagt agacagccag cgcgcgatct cgctgccgat caggtccggg ttgtcttctt
     3181 gcagcagatt cagacccggg ccgatgtcca cagccttgca gttaggcagg cttttggcca
     3241 ggcgagcggc ttcggccggt gggatcagaa cgcctggggt gccccagaac agcagcttcg
     3301 ggacagggga ctggtgcagc cagtccatgt attcttcgac cagcgcgacg atgttcgctg
     3361 gctcaccggc gattggcagc tcgtttggga agcgccacag tggctcgcgg tcaacaggat
     3421 tcaggaacgg ctcgcggtaa tggtccatct cgacttcagt cagcgggcgg acgacaccca
     3481 tcggcagcgt accctcgata aaaacgttct gatcgatgat cagcttgcgg ccgacgtcgg
     3541 tggtgcggaa ggcctggaag gtctcgcggg caaattctgg ccattcgtcc caggtcggga
     3601 tagggcggat gaactccata aatgcaatac ctttgacgcg ctctggattg cgcttggccc
     3661 agtggaaacc cagagcggag ccccagtcgt gaatgaccag gacgacctct tccagaccca
     3721 gggcttcgat gaaggcatcc atgaagcgga cgtggtcgtc gaagaaataa cccaggtctg
     3781 gtttgtcgga tttgcccata ccgatcaggt ctggagcaat gcagcgatgg gtcggtgcaa
     3841 catgcgggat gatgttgcgc cacacgtagg aggaggtcgg gttaccgtgc aggaacagca
     3901 caggggtgcc atcgcgcgga ccaacatcga cgtagtgcat gcgctcgccc aggacttcca
     3961 cataatgggg gtcgaatgga aagccagtac cgatttcgga tccatcgtta tcgctctgaa
     4021 agtacagatc ctcagtggtt ggctcgagag aaactctgga gatgtaggtg tatgttcgag
     4081 aaggggaacg ggggggttca gcttgggcag ctggatcatt taggaagtcc cagatgagga
     4141 ttgtgtcatc atgtgaacta ctgacaatct ggaattcatc aaactgtagt cgaaaaactc
     4201 ttccggaatg ctccacaagg gtccgtagac agagtgtccc tgcaggagca cgggggtcca
     4261 aagcagccac aagatcccac actttaattt ttccatcata ggccccactg actatcctct
     4321 tgttatcaaa tcgaatacaa cgcaccaatt cctcatggcc ttctaacact cgtaaacatg
     4381 caccacattc tatgtcccat aatctgatag tgttgtcaga tgagccactc actaccagcc
     4441 tgtccctgta ctgcaaacag gcaatgcctc gtttgtgtcc atttaaggtc cttacaaatt
     4501 cacaagtact tgtgttccat acctttatag ttctatcccc agatgcagaa acaatgtact
     4561 tgtcatcaaa gtctacaaca ttgacagcag ctcggtgtcc gaccagcacc ctccggaggg
     4621 taatgtcagt tggggaggcc atatcccata cagcaatgga acgatctttg gagcaggtca
     4681 ccatcatgcc attattgaaa cgcaagtgca gaactgcttc acaatggtga atcaacgtgt
     4741 ttagcatttc acctgtattt acatcccaca ctctgaccgt ggaatccgat gatcctgtta
     4801 tgatcactct ctcatcatac tggagacaga ggactgaacc tgtatggcct gtgagaattc
     4861 gcttgcattc caatgtgttt ttatcccaga tcttgattgt gttgtctcga aggccgctta
     4921 ctattttctg atcatcatac tgtaaacagt aaactccttt gcttgtttca cttcggcagt
     4981 gaattctctg taaactatgt cttccacatc tccaattaga ttctattgtc tcaatgtctt
     5041 gtataatttt aggataaagt gctctataaa aagagttggg aggagcattc ccgtcaggag
     5101 gtttgttttt gaataaatac tgtccccatc ctcttcgttc tgccaggcct ctccacagag
     5161 aatctgtcct gaccattctc tcgataagct tcttccacag catgccatca gaggtcactc
     5221 ggtaccattc cttgcacaca agttcagcag cacatagtga tttggcatcc aggtatgaca
     5281 gaatgttctc agcaatatga tccaatcccc gagctggcag agcagttatg aaatctctct
     5341 gcaacatggc gatcgctagc cctatagtga gtcgtattaa tactctagcc ttaagagctg
     5401 taattgaact gggagtggac acctgtggag agaaaggcaa agtggatgtc agtaagacca
     5461 ataggtgcct atcagaaacg caagagtctt ctctgtctcg acaagcccag tttctattgg
     5521 tctccttaaa cctgtcttgt aaccttgata cttacctgcc cagtgcctca cgaccaactt
     5581 ctgcagctta agttcgagac tgttgtgtca gaagcactga ctgcgttagc aatttaactg
     5641 tgataaacta ccgcaataaa gcttctagtg atctgacggt tcactaaacc agctctgctt
     5701 atatagacct cccaccgtac acgcctaccg cccatttgcg tcaacggggc ggggttatta
     5761 cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg
     5821 gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt ggtgtactgc
     5881 caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga
     5941 aagtcccgta aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg
     6001 tcaatagggg gcggacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
     6061 cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca
     6121 tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt
     6181 accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt
     6241 gattactatt aataactagt ggcgcgccat aatcctaggc tctcgtacga agtcagctga
     6301 taacgtcgac gtattgcggc cgcggatcat aatcagccat accacatttg tagaggtttt
     6361 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     6421 tgttgttgtt aacttgttta ttgcagc
//
