LOCUS       sequence-481208-        5406 bp    DNA     circular SYN 30-JUL-2025
DEFINITION  Express CRAF(RBD)-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 5406)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5406)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 30, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: NanoBRET Assay Vector, CRAF(RBD)-HaloTag
FEATURES             Location/Qualifiers
     source          1..5406
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    61..182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(72..91)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(297..330)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        340..387
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             419..1213
                     /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(473..492)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1083..1102
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(1371..1959)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1451..1470)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    2085..2133
                     /note="synthetic polyadenylation signal"
     CDS             complement(2157..2555)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      2552..2561
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2586..2943)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2600..2735
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        2752..2943
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     2900..2920
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    3022..3103
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        3336..3639
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3640..3843
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3794..3814
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          3979..4111
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4094..4113
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     4155..4174
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4155..4173
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4460..4480
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             4496..5380
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
ORIGIN
        1 ggcatgcaag ctgatttggc tgctgccacc gctgagcaag gccgcttcga gcagacatga
       61 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
      121 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
      181 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt
      241 ttttaagcaa gtaaaacctc tacaaatgtg gtaaaatctt aattaagaat tctaatataa
      301 cttcgtatag catacattat acgaagttat ggatccactg ttgacaatta atcatcggca
      361 tagtatatcg gcatagtata atacgacaag gtgaggaact aaacccagga ggcagatcat
      421 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
      481 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
      541 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
      601 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
      661 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
      721 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
      781 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
      841 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
      901 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
      961 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     1021 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     1081 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     1141 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     1201 cgagttcttc tgattcgaaa tgaccgacca agcgacgccc aaccggtatc agctcactca
     1261 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     1321 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     1381 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     1441 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     1501 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     1561 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     1621 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     1681 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     1741 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     1801 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     1861 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     1921 tgcaagcagc agattacgcg cagaaaaaaa ggatttcaag aagatccttt gatcttttct
     1981 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2041 tcaaaaagga tcttcaccta gatcctttta tagtccggaa tcgtcacaca aaaaaccaac
     2101 acacagatgt aatgaaaata aagatatttt attgcggcca tcgtgatggc tagagtttag
     2161 ccctcccaca cataaccaga gggcagcaat tcacgaatcc caactgccgt cggctgtcca
     2221 tcactgtcct tcactatggc tttgatccca ggatgcagat cgagaagcac ctgtcggcac
     2281 cgtccgcagg ggctcaagat gcccctgttc tcatttccga tcgcgacgat acaagtcagg
     2341 ttgccagctg ccgcagcagc agcagtgccc agcaccacga gttctgcaca aggtccccca
     2401 gtaaaatgat atacattgac accagtgaag atgcggccgt cgctagagag agctgcgctg
     2461 gcgacgctgt agtcttcaga gatggggatg ctgttgattg tagccgttgc tctttcaatg
     2521 agggtggatt cttcttgaga caaaggcttg gccatggtgg cgctagtgtc agaagaatcg
     2581 agctttttgc aaaagcctag gcctccaaaa aagcctcctc actacttctg gaatagctca
     2641 gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga
     2701 gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg
     2761 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
     2821 tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
     2881 gagcctgggg actttccaca ccctaactga cacacattcc acagctggtt ctttccgcct
     2941 cagaaggtac ctaaccaagt tcctctttca gaggttattt caggccatgg tgctgcgcaa
     3001 gacgcgttaa ttaagacgtc ggcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     3061 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     3121 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     3181 ctacaaatgt ggtatggctg attatgatcc gcggccgcaa tacgtcgacg ttatcagctg
     3241 acttcgtacg agagcctagg attatggcgc gccactagtt attaatagta atcaattacg
     3301 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     3361 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     3421 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     3481 gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat tgacgtcaat
     3541 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga ctttcctact
     3601 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     3661 accaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     3721 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaataac
     3781 cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     3841 gctggtttag tgaaccgtca gatcactaga agctttattg cggtagttta tcacagttaa
     3901 attgctaacg cagtcagtgc ttctgacaca acagtctcga acttaagctg cagaagttgg
     3961 tcgtgaggca ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag
     4021 aaactgggct tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt
     4081 actgacatcc actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct
     4141 taaggctaga gtattaatac gactcactat agggctagcg atcgccatgc cttctaagac
     4201 aagcaacact atccgtgttt tcttgccgaa caagcaaaga acagtggtca atgtgcgaaa
     4261 tggaatgagc ttgcatgact gccttatgaa agcactcaag gtgaggggcc tgcaaccaga
     4321 gtgctgtgca gtgttcagac ttctccacga acacaaaggt aaaaaagcac gcttagattg
     4381 gaatactgat gctgcgtctt tgattggaga agaacttcaa gtagatttcc tggatcatgt
     4441 ttctctcgag ccaaccactg aggatctgta ctttcagagc gataacgatg gatccgaaat
     4501 cggtactggc tttccattcg acccccatta tgtggaagtc ctgggcgagc gcatgcacta
     4561 cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta acccgacctc
     4621 ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg acccatcgct gcattgctcc
     4681 agacctgatc ggtatgggca aatccgacaa accagacctg ggttatttct tcgacgacca
     4741 cgtccgcttc atggatgcct tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat
     4801 tcacgactgg ggctccgctc tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa
     4861 aggtattgca tttatggagt tcatccgccc tatcccgacc tgggacgaat ggccagaatt
     4921 tgcccgcgag accttccagg ccttccgcac caccgacgtc ggccgcaagc tgatcatcga
     4981 tcagaacgtt tttatcgagg gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt
     5041 cgagatggac cattaccgcg agccgttcct gaatcctgtt gaccgcgagc cactgtggcg
     5101 cttcccaaac gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga
     5161 atacatggac tggctgcacc agtcccctgt cccgaagctg ctgttctggg gcaccccagg
     5221 cgttctgatc ccaccggccg aagccgctcg cctggccaaa agcctgccta actgcaaggc
     5281 tgtggacatc ggcccgggtc tgaatctgct gcaagaagac aacccggacc tgatcggcag
     5341 cgagatcgcg cgctggctgt ctactctgga gatttccggt taatagaatt ctagagtcga
     5401 cctgca
//
