LOCUS       sequence-471810-        8004 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express KSR2-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 8004)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8004)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: KSR2-HaloTag Fusion Vector
FEATURES             Location/Qualifiers
     source          1..8004
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    8..89
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        322..625
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        626..829
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     780..800
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          965..1097
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1080..1099
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1141..1160
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1141..1159
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4044..4064
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             4080..4964
                     /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    5051..5172
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5062..5081)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(5287..5320)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5330..5377
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5409..6203
                     /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(5463..5482)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6073..6092
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(6361..6949)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6441..6460)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    7075..7123
                     /note="synthetic polyadenylation signal"
     CDS             complement(7147..7545)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      7542..7551
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7576..7933)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7590..7725
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        7742..7933
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     7890..7910
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 gacgtcggca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
       61 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
      121 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
      181 tggctgatta tgatccgcgg ccgcaatacg tcgacgttat cagctgactt cgtacgagag
      241 cctaggatta tggcgcgcca ctagttatta atagtaatca attacggggt cattagttca
      301 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      361 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      421 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      481 acatcaagtg tatcatatgc caagtccgcc ccctattgac gtcaatgacg gtaaatggcc
      541 cgcctggcat tatgcccagt acatgacctt acgggacttt cctacttggc agtacatcta
      601 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacacca atgggcgtgg
      661 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      721 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aataaccccg ccccgttgac
      781 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa
      841 ccgtcagatc actagaagct ttattgcggt agtttatcac agttaaattg ctaacgcagt
      901 cagtgcttct gacacaacag tctcgaactt aagctgcaga agttggtcgt gaggcactgg
      961 gcaggtaagt atcaaggtta caagacaggt ttaaggagac caatagaaac tgggcttgtc
     1021 gagacagaga agactcttgc gtttctgata ggcacctatt ggtcttactg acatccactt
     1081 tgcctttctc tccacaggtg tccactccca gttcaattac agctcttaag gctagagtat
     1141 taatacgact cactataggg ctagcgatcg ccatggatga ggaaaacatg acgaaaagcg
     1201 aggagcagca gcctctgagt ttgcaaaaag ccttacagca gtgcgaactg gtccaaaaca
     1261 tgatagactt gagcatctcc aacctggaag ggcttaggac caaatgtgct acctccaacg
     1321 acctcacaca aaaagaaatc cggaccctgg agagcaagct ggtgaagtac ttcagccggc
     1381 agctgtcctg caaaaagaag gtagccttgc aggagcgcaa cgcggagctg gacggcttcc
     1441 cccagctacg gcactggttc cgaatcgtcg atgtgcgcaa ggaggtcctg gaggaaatct
     1501 cccccggcca gctgagcctg gaggacctct tggagatgac ggatgaacag gtgtgcgaga
     1561 ctgtggagaa atacggagcc aaccgggagg agtgtgcccg cctcaacgcc tccctctcct
     1621 gcctcaggaa tgtccacatg tcaggaggca acctttccaa acaagactgg accatccagt
     1681 ggcccacgac agagacgggg aaggagaaca atcccgtgtg ccccccggag cccaccccgt
     1741 ggatccgcac ccatctctcc cagagcccca gggtcccgtc caagtgcgtc cagcactatt
     1801 gtcacaccag ccccactccc ggggcccctg tgtacaccca cgtggacagg cttaccgtgg
     1861 acgcctaccc gggcttgtgc ccgcccccgc cactggagtc gggccaccgt tccctgcccc
     1921 catcgccccg gcagcggcac gcggtccgca ccccgccgcg cacccccaac atcgtcacca
     1981 ccgtgacccc gccgggcacg ccgcccatga ggaagaagaa caagctgaag cccccgggga
     2041 ccccaccgcc ctcctcccga aaactgatac acttgatccc gggattcacc gcgctgcatc
     2101 ggagcaaatc ccacgagttc cagctggggc accgcgtgga cgaggcccac acgcccaaag
     2161 ccaagaagaa gagcaaaccc ttgaacctca agatccacag cagcgtaggc agctgcgaga
     2221 acatcccctc tcagcagcgc tccccgctgc tgtccgagcg ctccctccgc tccttctttg
     2281 tgggacacgc acctttcctg ccttccaccc ctcctgttca cactgaggcc aacttctctg
     2341 caaacacact gtcagtgcca cgctggtccc cgcagatccc tcgcagagat ctcggcaact
     2401 ccatcaagca caggttttcc accaagtact ggatgtctca gacgtgcaca gtctgtggga
     2461 aagggatgct ttttggcctc aagtgtaaaa actgcaagtt aaagtgccac aacaaatgca
     2521 ccaaagaagc cccaccctgt catcttctga tcatccaccg aggagatcca gcaaggttag
     2581 tccggacaga gtccgttccg tgtgacatca acaaccctct acggaagcca cctcgctatt
     2641 cagacctgca catcagtcag acgctcccca aaaccaacaa aatcaacaag gaccacatcc
     2701 ctgtccctta ccagccagac tccagcagca acccctcctc cacgacgtcc tccacgccct
     2761 cctcgccagc accccccctc cctcctagtg ccacgccgcc ttctccccta cacccttccc
     2821 cacagtgcac acggcagcag aagaacttca acctgccagc atcccactac tacaaataca
     2881 agcagcagtt catcttccca gatgtggtgc cggtgccgga gacgccgacc cgggcgcccc
     2941 aggtcatcct gcatccggtg acctcgaatc caatcttgga aggaaatcca ttacttcaaa
     3001 ttgaagtgga gccaacgtcg gagaatgaag aggtccatga tgaggccgaa gagtcagagg
     3061 atgacttcga ggagatgaac ctgtccctcc tctcggcccg gagcttccca cgcaaggcca
     3121 gccagaccag catcttcctt caggagtggg acatcccctt tgagcagctg gagatcggcg
     3181 agctcattgg aaagggccgc tttgggcaag tgtaccacgg ccgctggcat ggcgaggtgg
     3241 ccatccggct gattgacatt gagagggaca acgaggacca gctcaaggcc ttcaagcggg
     3301 aggtgatggc ctacaggcag acacggcatg agaacgtggt gcttttcatg ggtgcctgca
     3361 tgagcccgcc tcacctggcc atcatcacca gcctctgtaa gggacggacg ctctattccg
     3421 ttgtgaggga tgccaaaatc gttttggatg tcaacaaaac caggcagatt gctcaagaaa
     3481 ttgtgaaggg catgggctac ctccacgcca agggaatcct acacaaggac ctcaagtcaa
     3541 agaacgtctt ctatgacaac ggcaaagtgg tcatcacgga ctttggactc ttcagcattt
     3601 ctggggtgct gcaggctggc aggcgggagg acaaactgcg catccagaat ggctggctat
     3661 gccacctggc accagagatc atccgccagc tgtcccccga cacagaggag gataagctcc
     3721 ccttctccaa gcactctgac gtctttgccc ttggcacaat ctggtatgaa ctccacgcca
     3781 gggaatggcc tttcaagacc caaccagcag aggcaataat ctggcaaatg ggcacaggca
     3841 tgaaacccaa cctcagccag attggcatgg gaaaagaaat ctcggacatt cttctcttct
     3901 gctgggcctt tgaacaagaa gagagaccta ccttcaccaa gctcatggac atgctggaga
     3961 aactgccaaa gcgaaaccgt cgcctgtctc accctggaca tttctggaag tctgcagagc
     4021 tggtttctct cgagccaacc actgaggatc tgtactttca gagcgataac gatggatccg
     4081 aaatcggtac tggctttcca ttcgaccccc attatgtgga agtcctgggc gagcgcatgc
     4141 actacgtcga tgttggtccg cgcgatggca cccctgtgct gttcctgcac ggtaacccga
     4201 cctcctccta cgtgtggcgc aacatcatcc cgcatgttgc accgacccat cgctgcattg
     4261 ctccagacct gatcggtatg ggcaaatccg acaaaccaga cctgggttat ttcttcgacg
     4321 accacgtccg cttcatggat gccttcatcg aagccctggg tctggaagag gtcgtcctgg
     4381 tcattcacga ctggggctcc gctctgggtt tccactgggc caagcgcaat ccagagcgcg
     4441 tcaaaggtat tgcatttatg gagttcatcc gccctatccc gacctgggac gaatggccag
     4501 aatttgcccg cgagaccttc caggccttcc gcaccaccga cgtcggccgc aagctgatca
     4561 tcgatcagaa cgtttttatc gagggtacgc tgccgatggg tgtcgtccgc ccgctgactg
     4621 aagtcgagat ggaccattac cgcgagccgt tcctgaatcc tgttgaccgc gagccactgt
     4681 ggcgcttccc aaacgagctg ccaatcgccg gtgagccagc gaacatcgtc gcgctggtcg
     4741 aagaatacat ggactggctg caccagtccc ctgtcccgaa gctgctgttc tggggcaccc
     4801 caggcgttct gatcccaccg gccgaagccg ctcgcctggc caaaagcctg cctaactgca
     4861 aggctgtgga catcggcccg ggtctgaatc tgctgcaaga agacaacccg gacctgatcg
     4921 gcagcgagat cgcgcgctgg ctgtctactc tggagatttc cggttaatag aattctagag
     4981 tcgacctgca ggcatgcaag ctgatttggc tgctgccacc gctgagcaag gccgcttcga
     5041 gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     5101 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     5161 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg
     5221 tgggaggttt ttttaagcaa gtaaaacctc tacaaatgtg gtaaaatctt aattaagaat
     5281 tctaatataa cttcgtatag catacattat acgaagttat ggatccactg ttgacaatta
     5341 atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact aaacccagga
     5401 ggcagatcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     5461 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     5521 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     5581 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     5641 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     5701 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     5761 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     5821 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     5881 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     5941 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     6001 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     6061 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     6121 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     6181 gccttcttga cgagttcttc tgattcgaaa tgaccgacca agcgacgccc aaccggtatc
     6241 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6301 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6361 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6421 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6481 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6541 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6601 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6661 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6721 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6781 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     6841 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6901 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatttcaag aagatccttt
     6961 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7021 catgagatta tcaaaaagga tcttcaccta gatcctttta tagtccggaa tcgtcacaca
     7081 aaaaaccaac acacagatgt aatgaaaata aagatatttt attgcggcca tcgtgatggc
     7141 tagagtttag ccctcccaca cataaccaga gggcagcaat tcacgaatcc caactgccgt
     7201 cggctgtcca tcactgtcct tcactatggc tttgatccca ggatgcagat cgagaagcac
     7261 ctgtcggcac cgtccgcagg ggctcaagat gcccctgttc tcatttccga tcgcgacgat
     7321 acaagtcagg ttgccagctg ccgcagcagc agcagtgccc agcaccacga gttctgcaca
     7381 aggtccccca gtaaaatgat atacattgac accagtgaag atgcggccgt cgctagagag
     7441 agctgcgctg gcgacgctgt agtcttcaga gatggggatg ctgttgattg tagccgttgc
     7501 tctttcaatg agggtggatt cttcttgaga caaaggcttg gccatggtgg cgctagtgtc
     7561 agaagaatcg agctttttgc aaaagcctag gcctccaaaa aagcctcctc actacttctg
     7621 gaatagctca gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc
     7681 atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg
     7741 cgggactatg gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga
     7801 gcctggggac tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct
     7861 gcctgctggg gagcctgggg actttccaca ccctaactga cacacattcc acagctggtt
     7921 ctttccgcct cagaaggtac ctaaccaagt tcctctttca gaggttattt caggccatgg
     7981 tgctgcgcaa gacgcgttaa ttaa
//
