LOCUS       sequence-471867-        5826 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  Express hCHMP4B-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 5826)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 5826)
  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: hCHMP4B-HaloTag Vector
FEATURES             Location/Qualifiers
     source          1..5826
                     /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             1861..1881
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             1897..2781
                     /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    2868..2989
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2879..2898)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3104..3137)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3147..3194
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3226..4020
                     /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(3280..3299)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3890..3909
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4178..4766)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4258..4277)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4892..4940
                     /note="synthetic polyadenylation signal"
     CDS             complement(4964..5362)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5359..5368
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5393..5750)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5407..5542
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5559..5750
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5707..5727
                     /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 tcggtgttcg ggaagctgtt cggggctgga
     1201 gggggtaagg ccggcaaggg cggcccgacc ccccaggagg ccatccagcg gctgcgggac
     1261 acggaagaga tgttaagcaa gaaacaggag ttcctggaga agaaaatcga gcaggagctg
     1321 acggccgcca agaagcacgg caccaaaaac aagcgcgcgg ccctccaggc actgaagcgt
     1381 aagaagaggt atgagaagca gctggcgcag atcgacggca cattatcaac catcgagttc
     1441 cagcgggagg ccctggagaa tgccaacacc aacaccgagg tgctcaagaa catgggctat
     1501 gccgccaagg ccatgaaggc ggcccatgac aacatggaca tcgataaagt tgatgagtta
     1561 atgcaggaca ttgctgacca gcaagaactt gcagaggaga tttcaacagc aatttcgaaa
     1621 cctgtagggt ttggagaaga gtttgacgag gatgagctca tggcggaatt agaagaacta
     1681 gaacaggagg aactagacaa gaatttgctg gaaatcagtg gacccgaaac agtccctcta
     1741 ccaaatgttc cctctatagc cctaccatca aaacccgcca agaagaaaga agaggaggac
     1801 gacgacatga aggaattgga gaactgggct ggatccatgg tttctctcga gccaaccact
     1861 gaggatctgt actttcagag cgataacgat ggatccgaaa tcggtactgg ctttccattc
     1921 gacccccatt atgtggaagt cctgggcgag cgcatgcact acgtcgatgt tggtccgcgc
     1981 gatggcaccc ctgtgctgtt cctgcacggt aacccgacct cctcctacgt gtggcgcaac
     2041 atcatcccgc atgttgcacc gacccatcgc tgcattgctc cagacctgat cggtatgggc
     2101 aaatccgaca aaccagacct gggttatttc ttcgacgacc acgtccgctt catggatgcc
     2161 ttcatcgaag ccctgggtct ggaagaggtc gtcctggtca ttcacgactg gggctccgct
     2221 ctgggtttcc actgggccaa gcgcaatcca gagcgcgtca aaggtattgc atttatggag
     2281 ttcatccgcc ctatcccgac ctgggacgaa tggccagaat ttgcccgcga gaccttccag
     2341 gccttccgca ccaccgacgt cggccgcaag ctgatcatcg atcagaacgt ttttatcgag
     2401 ggtacgctgc cgatgggtgt cgtccgcccg ctgactgaag tcgagatgga ccattaccgc
     2461 gagccgttcc tgaatcctgt tgaccgcgag ccactgtggc gcttcccaaa cgagctgcca
     2521 atcgccggtg agccagcgaa catcgtcgcg ctggtcgaag aatacatgga ctggctgcac
     2581 cagtcccctg tcccgaagct gctgttctgg ggcaccccag gcgttctgat cccaccggcc
     2641 gaagccgctc gcctggccaa aagcctgcct aactgcaagg ctgtggacat cggcccgggt
     2701 ctgaatctgc tgcaagaaga caacccggac ctgatcggca gcgagatcgc gcgctggctg
     2761 tctactctgg agatttccgg ttaatagaat tctagagtcg acctgcaggc atgcaagctg
     2821 atttggctgc tgccaccgct gagcaaggcc gcttcgagca gacatgataa gatacattga
     2881 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     2941 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     3001 ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt taagcaagta
     3061 aaacctctac aaatgtggta aaatcttaat taagaattct aatataactt cgtatagcat
     3121 acattatacg aagttatgga tccactgttg acaattaatc atcggcatag tatatcggca
     3181 tagtataata cgacaaggtg aggaactaaa cccaggaggc agatcatgat tgaacaagat
     3241 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3301 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3361 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     3421 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3481 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3541 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3601 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3661 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     3721 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     3781 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     3841 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     3901 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     3961 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4021 ttcgaaatga ccgaccaagc gacgcccaac cggtatcagc tcactcaaag gcggtaatac
     4081 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4141 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4201 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4261 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4321 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4381 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4441 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4501 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4561 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     4621 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4681 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4741 ttacgcgcag aaaaaaagga tttcaagaag atcctttgat cttttctacg gggtctgacg
     4801 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4861 tcacctagat ccttttatag tccggaatcg tcacacaaaa aaccaacaca cagatgtaat
     4921 gaaaataaag atattttatt gcggccatcg tgatggctag agtttagccc tcccacacat
     4981 aaccagaggg cagcaattca cgaatcccaa ctgccgtcgg ctgtccatca ctgtccttca
     5041 ctatggcttt gatcccagga tgcagatcga gaagcacctg tcggcaccgt ccgcaggggc
     5101 tcaagatgcc cctgttctca tttccgatcg cgacgataca agtcaggttg ccagctgccg
     5161 cagcagcagc agtgcccagc accacgagtt ctgcacaagg tcccccagta aaatgatata
     5221 cattgacacc agtgaagatg cggccgtcgc tagagagagc tgcgctggcg acgctgtagt
     5281 cttcagagat ggggatgctg ttgattgtag ccgttgctct ttcaatgagg gtggattctt
     5341 cttgagacaa aggcttggcc atggtggcgc tagtgtcaga agaatcgagc tttttgcaaa
     5401 agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gccgaggcgg
     5461 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac
     5521 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat
     5581 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg
     5641 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
     5701 ttccacaccc taactgacac acattccaca gctggttctt tccgcctcag aaggtaccta
     5761 accaagttcc tctttcagag gttatttcag gccatggtgc tgcgcaagac gcgttaatta
     5821 agacgt
//
