LOCUS       sequence-471811-        7923 bp    DNA     circular SYN 06-JUN-2025
DEFINITION  Express KSR1-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 7923)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 7923)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: KSR1-HaloTag Fusion Vector
FEATURES             Location/Qualifiers
     source          1..7923
                     /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             3963..3983
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             3999..4883
                     /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    4970..5091
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4981..5000)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(5206..5239)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5249..5296
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5328..6122
                     /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(5382..5401)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5992..6011
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(6280..6868)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6360..6379)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    6994..7042
                     /note="synthetic polyadenylation signal"
     CDS             complement(7066..7464)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      7461..7470
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7495..7852)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7509..7644
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        7661..7852
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     7809..7829
                     /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 ccatggatag agcagcgctg cgcgcggcgg
     1201 cgatgggaga gaagaaggag ggcggtggcg ggggggatgc ggcggccgcg gagggaggcg
     1261 caggggccgc ggccagccgg gcgctgcagc agtgcgggca gctccagaag ctcatcgaca
     1321 tctccatcgg cagcctgcgc gggctgcgca ccaagtgcgc tgtgtccaac gacctcaccc
     1381 agcaggagat acggaccctg gaggcgaagc tggtccgtta catttgtaag cagaggcagt
     1441 gcaagctgag cgtggctccc ggtgagagga ccccagagct caacagctac ccccgcttca
     1501 gcgactggct gtacactttc aacgtgaggc cggaggtggt gcaggagatc ccccgagacc
     1561 tcacgctgga tgccctgctg gagatgaatg aggccaaggt gaaggagacg ctgcggcgct
     1621 gtggggccag cggggatgag tgtggccgtc tgcagtatgc cctcacctgc ctgcggaagg
     1681 tgacaggcct gggaggggag cacaaggagg actccagttg gagttcattg gatgcgcggc
     1741 gggaaagtgg ctcagggcct tccacggaca ccctctcagc agccagcctg ccctggcccc
     1801 cagggagctc ccagctgggc agagcaggca acagcgccca gggcccacgc tccatctccg
     1861 tgtcagctct gcccgcctca gactccccca cccccagctt cagtgagggc ctctcagaca
     1921 cctgtattcc cctgcacgcc agcggccggc tgaccccccg tgccctgcac agcttcatca
     1981 ccccgcccac cacaccccag ctgcgacggc acaccaagct gaagccacca cggacgcccc
     2041 ccccacccag ccgcaaggtc ttccagctgc tgcccagctt ccccacactc acccggagca
     2101 agtcccatga gtctcagctg gggaaccgca ttgatgacgt ctcctcgatg aggtttgatc
     2161 tctcgcatgg atccccacag atggtacgga gggatatcgg gctgtcggtg acgcacaggt
     2221 tctccaccaa gtcctggctg tcgcaggtct gccacgtgtg ccagaagagc atgatatttg
     2281 gagtgaagtg caagcattgc aggttgaagt gtcacaacaa atgtaccaaa gaagcccctg
     2341 cctgtagaat atccttcctg ccactaactc ggcttcggag gacagaatct gtcccctcgg
     2401 acatcaacaa cccggtggac agagcagccg aaccccattt tggaaccctc cccaaagcac
     2461 tgacaaagaa ggagcaccct ccggccatga atcacctgga ctccagcagc aacccttcct
     2521 ccaccacctc ctccacaccc tcctcaccgg cgcccttccc gacatcatcc aacccatcca
     2581 gcgccaccac gccccccaac ccctcacctg gccagcggga cagcaggttc aacttcccag
     2641 ctgcctactt cattcatcat agacagcagt ttatctttcc agtgccatct gctggccatt
     2701 gctggaaatg cctccttatt gcagaaagtt taaaggaaaa cgctttcaac atttcagcct
     2761 ttgcacacgc agccccgctc cctgaagctg ccgacggtac ccggctcgat gaccagccga
     2821 aagcagatgt gttggaagct cacgaagcgg aggctgagga gccagaggct ggcaagtcag
     2881 aggcagaaga cgatgaggac gaggtggacg acttgccgag ctctcgccgg ccctggcggg
     2941 gccccatctc tcgcaaggcc agccagacca gcgtgtacct gcaggagtgg gacatcccct
     3001 tcgagcaggt agagctgggc gagcccatcg ggcagggccg ctggggccgg gtgcaccgcg
     3061 gccgctggca tggcgaggtg gccattcgcc tgctggagat ggacggccac aaccaggacc
     3121 acctgaagct cttcaagaaa gaggtgatga actaccggca gacgcggcat gagaacgtgg
     3181 tgctcttcat gggggcctgc atgaacccgc cccacctggc cattatcacc agcttctgca
     3241 aggggcggac gttgcactcg tttgtgaggg accccaagac gtctctggac atcaacaaga
     3301 cgaggcaaat cgctcaggag atcatcaagg gcatgggata tcttcatgcc aagggcatcg
     3361 tacacaaaga tctcaaatct aagaacgtct tctatgacaa cggcaaggtg gtcatcacag
     3421 acttcgggct gtttgggatc tcaggcgtgg tccgagaggg acggcgtgag aaccagctaa
     3481 agctgtccca cgactggctg tgctatctgg cccctgagat tgtacgcgag atgacccccg
     3541 ggaaggacga ggatcagctg ccattctcca aagctgctga tgtctatgca tttgggactg
     3601 tttggtatga gctgcaagca agagactggc ccttgaagaa ccaggctgca gaggcatcca
     3661 tctggcagat tggaagcggg gaaggaatga agcgtgtcct gacttctgtc agcttgggga
     3721 aggaagtcag tgagatcctg tcggcctgct gggctttcga cctgcaggag agacccagct
     3781 tcagcctgct gatggacatg ctggagaaac ttcccaagct gaaccggcgg ctctcccacc
     3841 ctggacactt ctggaagtca gctgacatta acagcagcaa agttgtaccc cggtttgaaa
     3901 ggtttggctt gggcgtcctg gagtccagta atccaaagat ggtttctctc gagccaacca
     3961 ctgaggatct gtactttcag agcgataacg atggatccga aatcggtact ggctttccat
     4021 tcgaccccca ttatgtggaa gtcctgggcg agcgcatgca ctacgtcgat gttggtccgc
     4081 gcgatggcac ccctgtgctg ttcctgcacg gtaacccgac ctcctcctac gtgtggcgca
     4141 acatcatccc gcatgttgca ccgacccatc gctgcattgc tccagacctg atcggtatgg
     4201 gcaaatccga caaaccagac ctgggttatt tcttcgacga ccacgtccgc ttcatggatg
     4261 ccttcatcga agccctgggt ctggaagagg tcgtcctggt cattcacgac tggggctccg
     4321 ctctgggttt ccactgggcc aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg
     4381 agttcatccg ccctatcccg acctgggacg aatggccaga atttgcccgc gagaccttcc
     4441 aggccttccg caccaccgac gtcggccgca agctgatcat cgatcagaac gtttttatcg
     4501 agggtacgct gccgatgggt gtcgtccgcc cgctgactga agtcgagatg gaccattacc
     4561 gcgagccgtt cctgaatcct gttgaccgcg agccactgtg gcgcttccca aacgagctgc
     4621 caatcgccgg tgagccagcg aacatcgtcg cgctggtcga agaatacatg gactggctgc
     4681 accagtcccc tgtcccgaag ctgctgttct ggggcacccc aggcgttctg atcccaccgg
     4741 ccgaagccgc tcgcctggcc aaaagcctgc ctaactgcaa ggctgtggac atcggcccgg
     4801 gtctgaatct gctgcaagaa gacaacccgg acctgatcgg cagcgagatc gcgcgctggc
     4861 tgtctactct ggagatttcc ggttaataga attctagagt cgacctgcag gcatgcaagc
     4921 tgatttggct gctgccaccg ctgagcaagg ccgcttcgag cagacatgat aagatacatt
     4981 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     5041 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     5101 aattgcattc attttatgtt tcaggttcag ggggagatgt gggaggtttt tttaagcaag
     5161 taaaacctct acaaatgtgg taaaatctta attaagaatt ctaatataac ttcgtatagc
     5221 atacattata cgaagttatg gatccactgt tgacaattaa tcatcggcat agtatatcgg
     5281 catagtataa tacgacaagg tgaggaacta aacccaggag gcagatcatg attgaacaag
     5341 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     5401 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     5461 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     5521 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     5581 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     5641 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     5701 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     5761 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     5821 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     5881 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     5941 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     6001 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     6061 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     6121 gattcgaaat gaccgaccaa gcgacgccca accggtatca gctcactcaa aggcggtaat
     6181 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     6241 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6301 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6361 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6421 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6481 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6541 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6601 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6661 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6721 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6781 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6841 gattacgcgc agaaaaaaag gatttcaaga agatcctttg atcttttcta cggggtctga
     6901 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6961 cttcacctag atccttttat agtccggaat cgtcacacaa aaaaccaaca cacagatgta
     7021 atgaaaataa agatatttta ttgcggccat cgtgatggct agagtttagc cctcccacac
     7081 ataaccagag ggcagcaatt cacgaatccc aactgccgtc ggctgtccat cactgtcctt
     7141 cactatggct ttgatcccag gatgcagatc gagaagcacc tgtcggcacc gtccgcaggg
     7201 gctcaagatg cccctgttct catttccgat cgcgacgata caagtcaggt tgccagctgc
     7261 cgcagcagca gcagtgccca gcaccacgag ttctgcacaa ggtcccccag taaaatgata
     7321 tacattgaca ccagtgaaga tgcggccgtc gctagagaga gctgcgctgg cgacgctgta
     7381 gtcttcagag atggggatgc tgttgattgt agccgttgct ctttcaatga gggtggattc
     7441 ttcttgagac aaaggcttgg ccatggtggc gctagtgtca gaagaatcga gctttttgca
     7501 aaagcctagg cctccaaaaa agcctcctca ctacttctgg aatagctcag aggccgaggc
     7561 ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca tggggcggag aatgggcgga
     7621 actgggcgga gttaggggcg ggatgggcgg agttaggggc gggactatgg ttgctgacta
     7681 attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact ttccacacct
     7741 ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga
     7801 ctttccacac cctaactgac acacattcca cagctggttc tttccgcctc agaaggtacc
     7861 taaccaagtt cctctttcag aggttatttc aggccatggt gctgcgcaag acgcgttaat
     7921 taa
//
