LOCUS       sequence-482026-        6430 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Express HaloTag-CALR 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 6430)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6430)
  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            "Unpublished"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: HaloTag-CALR Fusion Vector
FEATURES             Location/Qualifiers
     source          1..6430
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        156..513
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        156..347
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(179..199)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      364..499
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      538..547
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             544..942
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    966..1014
                     /note="synthetic polyadenylation signal"
     rep_origin      1140..1728
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1629..1648
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1886..2680)
                     /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(1997..2016)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2607..2626
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2712..2759)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     protein_bind    2769..2802
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2917..3038
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3008..3027
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(4417..4437)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             complement(4450..5340)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     regulatory      5337..5346
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(5355..5374)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5356..5374)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5416..5435)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          5418..5550
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        complement(5686..5889)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5715..5735)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5890..6193
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     polyA_signal    join(6426..6430,1..77)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
       61 agcatttttt tcactgccga cgtcttaatt aacgcgtctt gcgcagcacc atggcctgaa
      121 ataacctctg aaagaggaac ttggttaggt accttctgag gcggaaagaa ccagctgtgg
      181 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
      241 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
      301 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
      361 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
      421 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
      481 ggaggctttt ttggaggcct aggcttttgc aaaaagctcg attcttctga cactagcgcc
      541 accatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca
      601 atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac
      661 ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc
      721 gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc
      781 ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat
      841 ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg
      901 attcgtgaat tgctgccctc tggttatgtg tgggagggct aaactctagc catcacgatg
      961 gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgacgatt
     1021 ccggactata aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     1081 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1141 tgaaatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1201 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1261 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     1321 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     1381 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     1441 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1501 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     1561 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     1621 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     1681 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     1741 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     1801 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccggttgg
     1861 gcgtcgcttg gtcggtcatt tcgaatcaga agaactcgtc aagaaggcga tagaaggcga
     1921 tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc
     1981 cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca
     2041 cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg
     2101 gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga
     2161 gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat
     2221 cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt
     2281 cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg
     2341 atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca
     2401 atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc
     2461 ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg
     2521 acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg
     2581 catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag
     2641 cggccggaga acctgcgtgc aatccatctt gttcaatcat gatctgcctc ctgggtttag
     2701 ttcctcacct tgtcgtatta tactatgccg atatactatg ccgatgatta attgtcaaca
     2761 gtggatccat aacttcgtat aatgtatgct atacgaagtt atattagaat tcttaattaa
     2821 gattttacca catttgtaga ggttttactt gcttaaaaaa acctcccaca tctccccctg
     2881 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     2941 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     3001 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgctc gaagcggcct
     3061 tgctcagcgg tggcagcagc caaatcagct tgcatgcctg caggtcgact ctagaggatc
     3121 cccgggtacc gagcccgaat tcgtttaaac cagctcgtcc ttggcctggc cggggacatc
     3181 ttcctcctca tcttcctcct tgtcctcctc atcctcctca tcctcatctt tgtcctcatc
     3241 atcctccttg tcctctgcct cctcctcctc tttgcgtttc ttgtcttctt cctcctcctt
     3301 aagcctctgc tcctcgtcct gtttgtcctt catttgtttc tctgctgcct ttgttacgcc
     3361 ccacgtctcg ttgccaaact cctcagcgta tgcctcatcg ttggtgatga ggaagttgtc
     3421 aaagatggtg ccagacttga cctgccagag gtccaggccc agcacgccaa agttatcata
     3481 ggcatagata ctgggatcgg gagaatactc ggggttgtca atttctgggt ggatccaagt
     3541 gcccttgtaa tctgggttgt cgatctgccg gggcttccac tcacccttgt actcagggtt
     3601 ctgaatcact gggggttccc actctccgtc catctcttca tcccagtcct cgggcttctt
     3661 agcatcaggg tcagggatat gctcgggctt gtcccagtcc tcaggcttgg agtctgtggg
     3721 atcatcgatc ttggcccgct catcccagtc ttccggtttt gaagcatcag gatcctttat
     3781 cttcttgggt ggcaggaagt cccaatcgtc ttccaaggag ccggactcca cctggctgtt
     3841 gtcaatcttc acctcatagg tgttgtctgg ccgcacaatc agtgtgtaca ggtgtgtaaa
     3901 ctcatcatcc ttgcaacgga tgtccttgtt gatcagcacg ttcttgccct tgtagttgaa
     3961 gatgacatga accttcttgg tgccagggcc acagatgtcg ggaccaaaca tgatgttgta
     4021 ttctgagtct ccgtgcatgt ctgtctggtc caaactatta ggaaacagct tcacatagcc
     4081 gcccccacag tcgatgttct gctcatgttt caccgtgaac tgcaccacca gcgtctggcc
     4141 tttgttgctg aaaggctcga aactggccga cagagcataa aagcgtgcat cctggcttgt
     4201 ctgcaaacct ttatctttct cctcgtcacc gtagaacttg ccggaactga gaacgaattt
     4261 gccaaaatct gacttgtgtt tggattcgat ccagcgggaa gtccacccgt ctccgtccag
     4321 aaactgctcc ttgaagtaga cggcaggctc ggcgacggcc aggccgagga ggccgagcag
     4381 cagcggcacg gatagcagca tggcgatcgc gttatcgctc tgaaagtaca gatcctcagt
     4441 ggttggctcg ccggaaatct cgagcgtcga cagccagcgc gcgatctcgc tgccgatcag
     4501 gtccgggttg tcttcttgca gcagattcag acccgggccg atgtccacag ccttgcagtt
     4561 aggcaggctt ttggccaggc gagcggcttc ggccggtggg atcagaacgc ctggggtgcc
     4621 ccagaacagc agcttcggga caggggactg gtgcagccag tccatgtatt cttcgaccag
     4681 cgcgacgatg ttcgctggct caccggcgat tggcagctcg tttgggaagc gccacagtgg
     4741 ctcgcggtca acaggattca ggaacggctc gcggtaatgg tccatctcga cttcagtcag
     4801 cgggcggacg acacccatcg gcagcgtacc ctcgataaaa acgttctgat cgatgatcag
     4861 cttgcggccg acgtcggtgg tgcggaaggc ctggaaggtc tcgcgggcaa attctggcca
     4921 ttcgtcccag gtcgggatag ggcggatgaa ctccataaat gcaatacctt tgacgcgctc
     4981 tggattgcgc ttggcccagt ggaaacccag agcggagccc cagtcgtgaa tgaccaggac
     5041 gacctcttcc agacccaggg cttcgatgaa ggcatccatg aagcggacgt ggtcgtcgaa
     5101 gaaataaccc aggtctggtt tgtcggattt gcccataccg atcaggtctg gagcaatgca
     5161 gcgatgggtc ggtgcaacat gcgggatgat gttgcgccac acgtaggagg aggtcgggtt
     5221 accgtgcagg aacagcacag gggtgccatc gcgcggacca acatcgacgt agtgcatgcg
     5281 ctcgcccagg acttccacat aatgggggtc gaatggaaag ccagtaccga tttctgccat
     5341 ggtggctttg ctagccctat agtgagtcgt attaatactc tagccttaag agctgtaatt
     5401 gaactgggag tggacacctg tggagagaaa ggcaaagtgg atgtcagtaa gaccaatagg
     5461 tgcctatcag aaacgcaaga gtcttctctg tctcgacaag cccagtttct attggtctcc
     5521 ttaaacctgt cttgtaacct tgatacttac ctgcccagtg cctcacgacc aacttctgca
     5581 gcttaagttc gagactgttg tgtcagaagc actgactgcg ttagcaattt aactgtgata
     5641 aactaccgca ataaagcttc tagtgatctg acggttcact aaaccagctc tgcttatata
     5701 gacctcccac cgtacacgcc taccgcccat ttgcgtcaac ggggcggggt tattacgaca
     5761 ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg
     5821 gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattggtgt actgccaaaa
     5881 ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc
     5941 ccgtaaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat
     6001 agggggcgga cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa
     6061 atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt
     6121 cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt
     6181 aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta
     6241 ctattaataa ctagtggcgc gccataatcc taggctctcg tacgaagtca gctgataacg
     6301 tcgacgtatt gcggccgcgg atcataatca gccataccac atttgtagag gttttacttg
     6361 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     6421 ttgttaactt
//
