LOCUS       sequence-479076-        7120 bp    DNA     circular SYN 21-JUL-2025
DEFINITION  Express HiBiT-RAF1 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 7120)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7120)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 21, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: HiBiT-RAF1 Fusion Vector
FEATURES             Location/Qualifiers
     source          1..7120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1446..1466)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             complement(1479..2369)
                     /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      2366..2375
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2384..2403)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2385..2403)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2445..2464)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          2447..2579
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        complement(2715..2918)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2744..2764)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2919..3298
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    3489..3772
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     rep_origin      3888..4476
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4377..4396
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4715..4763
                     /note="synthetic polyadenylation signal"
     CDS             complement(4827..5621)
                     /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(4938..4957)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5548..5567
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5653..5700)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5715..6072)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5729..5864
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5881..6072
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6029..6049
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    6317..6438
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6354..6373)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6408..6427
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6475..6493
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 tggagaaata tattgttgga tttcatgtct ctatggatga tgttctttgc atgcaaatag
       61 tccattccct gagccgtctg ccgggcaatg tcaattagct ggaacatctg aaacttggtc
      121 tcctggacat gcaggtgttt gtagaggctg ctgccctcgc accactgggt cacaattgcc
      181 aggttgtcct ttgtcatgta ccccatgaaa agcagaatgt tcacatgccg tgttttgcgc
      241 agaacagcca cctcattcct gaaggcctgg aattgctctg gggttgggtc gacaaccttt
      301 aggatcttta ctgcaacatc tccgtgccat ttacccttat aaacagttcc aaaagagcct
      361 gacccaatcc gagtggacag catcacttca ctggcttcta tttcccaata atagcttgaa
      421 tctctctgtc cacgaggcct aattttgttt ttctcctggg tcccagatac tggtgcccgc
      481 tctctttgtg ctggcacggg ggttttcggc tgtgaccagc ctgttgggct cagattgttg
      541 gggctactgg acagggctga aggtgaggct gattcgctgt gacttcgaat tgcatcctca
      601 atcatcctgc tgtccacagg cagggtggtg ctgaccatgt ggacattagg tgtggatgtc
      661 gacctctgcc tctgggagag ggaaccttca gatgagggac tggaggtgtt aaaggtgaag
      721 gcgtgaggtg tagaatatct gtgctgagaa ctaacaggca tcctggaaac agactctcgc
      781 atacgacgca tagtcaaaga aggtagtgct gggactccac tatcaccaat agtggaattt
      841 ggaaacaata agagttgtct gatgttactc cagtccacac acatagtagg tactttggtg
      901 ctacagtgct catgaaattt gtagccacaa gtctgacatc gaaatccatt gagcaggaat
      961 ttctgacaga tgtcacagaa ggcaagcttc aggaacgtct tccgagcaaa gttgtgtgtt
     1021 gtgaggggaa catgatccag gaaatctact tgaagttctt ctccaatcaa agacgcagca
     1081 tcagtattcc aatctaagcg tgctttttta cctttgtgtt cgtggagaag tctgaacact
     1141 gcacagcact ctggttgcag gcccctcacc ttgagtgctt tcataaggca gtcatgcaag
     1201 ctcattccat ttcgcacatt gaccactgtt ctttgcttgt tcggcaagaa aacacggata
     1261 gtgttgcttg tcttagaagg atctgtgagt ttgccatcat ctgatgcccg gcgctgatag
     1321 ccaaactgct gaactattgt aggagagatg cagctggagc catcaaacac ggcatctttg
     1381 aatccaaaac cattgctgat cgtcttccaa gctccctgta tgtgctccat ggcgatcgcg
     1441 ttatcgctct gaaagtacag atcctcagtg gttggctcgc cggaaatctc gagcgtcgac
     1501 agccagcgcg cgatctcgct gccgatcagg tccgggttgt cttcttgcag cagattcaga
     1561 cccgggccga tgtccacagc cttgcagtta ggcaggcttt tggccaggcg agcggcttcg
     1621 gccggtggga tcagaacgcc tggggtgccc cagaacagca gcttcgggac aggggactgg
     1681 tgcagccagt ccatgtattc ttcgaccagc gcgacgatgt tcgctggctc accggcgatt
     1741 ggcagctcgt ttgggaagcg ccacagtggc tcgcggtcaa caggattcag gaacggctcg
     1801 cggtaatggt ccatctcgac ttcagtcagc gggcggacga cacccatcgg cagcgtaccc
     1861 tcgataaaaa cgttctgatc gatgatcagc ttgcggccga cgtcggtggt gcggaaggcc
     1921 tggaaggtct cgcgggcaaa ttctggccat tcgtcccagg tcgggatagg gcggatgaac
     1981 tccataaatg caataccttt gacgcgctct ggattgcgct tggcccagtg gaaacccaga
     2041 gcggagcccc agtcgtgaat gaccaggacg acctcttcca gacccagggc ttcgatgaag
     2101 gcatccatga agcggacgtg gtcgtcgaag aaataaccca ggtctggttt gtcggatttg
     2161 cccataccga tcaggtctgg agcaatgcag cgatgggtcg gtgcaacatg cgggatgatg
     2221 ttgcgccaca cgtaggagga ggtcgggtta ccgtgcagga acagcacagg ggtgccatcg
     2281 cgcggaccaa catcgacgta gtgcatgcgc tcgcccagga cttccacata atgggggtcg
     2341 aatggaaagc cagtaccgat ttctgccatg gtggctttgc tagccctata gtgagtcgta
     2401 ttaatactct agccttaaga gctgtaattg aactgggagt ggacacctgt ggagagaaag
     2461 gcaaagtgga tgtcagtaag accaataggt gcctatcaga aacgcaagag tcttctctgt
     2521 ctcgacaagc ccagtttcta ttggtctcct taaacctgtc ttgtaacctt gatacttacc
     2581 tgcccagtgc ctcacgacca acttctgcag cttaagttcg agactgttgt gtcagaagca
     2641 ctgactgcgt tagcaattta actgtgataa actaccgcaa taaagcttct agtgatctga
     2701 cggttcacta aaccagctct gcttatatag acctcccacc gtacacgcct accgcccatt
     2761 tgcgtcaacg gggcggggtt attacgacat tttggaaagt cccgttgatt ttggtgccaa
     2821 aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc
     2881 tatccacgcc cattggtgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat
     2941 acgtagatgt actgccaagt aggaaagtcc cgtaaggtca tgtactgggc ataatgccag
     3001 gcgggccatt taccgtcatt gacgtcaata gggggcggac ttggcatatg atacacttga
     3061 tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc
     3121 cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg
     3181 ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg
     3241 ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa tcaatgccaa
     3301 catggcggtc atattggaca tgagccaata taaatgtaca tattatgata tagatacaac
     3361 gtatgcaatg gccaatagcc aatattgatt tatgctatat aaccaatgaa taatatggct
     3421 aatggccaat attgaagatc tcgatcctct acgccggacg catcgtggcc ggagtactgc
     3481 ggtttatctc gatggctacg agggcagaca gtaagtggat ttaccataat cccttaattg
     3541 tacgcaccgc taaaacgcgt tcagcgcgat cacggcagca gacaggtaaa aatggcaaca
     3601 aaccacccga aaaactgccg caatcgcgcc tgataaattt taaccgtatg aatacctatg
     3661 caaccagagg gtacaggcca cattaccccc acttaatcca ctgaagctgc catttttcat
     3721 ggtttcacca tcccagcgaa gggccatcca gcgtgcgttc ctgtatttcc ggactataaa
     3781 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     3841 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg aaatcctttt
     3901 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3961 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4021 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4081 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4141 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4201 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4261 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4321 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4381 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4441 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4501 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4561 tctgtggata accgtattac cgcctttgag tgagctgata ccgggtagaa agccagtccg
     4621 cagaaacggt gctgaccccg gatgaatgtc agctactggg ctatctggac aagggaaaac
     4681 gcaagcgcaa agaggatcct tatcgctatc gattcacaca aaaaaccaac acacagatgt
     4741 aatgaaaata aagatatttt attgcggcca tcgtgatggc aggttgggcg tcgcttggtc
     4801 ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg
     4861 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     4921 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     4981 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     5041 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     5101 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     5161 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     5221 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     5281 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     5341 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     5401 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     5461 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     5521 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     5581 gcggccggag aacctgcgtg caatccatct tgttcaatca tgatctgcct cctgggttta
     5641 gttcctcacc ttgtcgtatt atactatgcc gatatactat gccgatgatt aattgtcaac
     5701 agttaattaa gctttttgca aaagcctagg cctccaaaaa agcctcctca ctacttctgg
     5761 aatagctcag aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca
     5821 tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc
     5881 gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
     5941 cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg catacttctg
     6001 cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca cagctggttc
     6061 tttccgcctc agaaggtacc taaccaagtt cctctttcag aggttatttc aggccatggt
     6121 gctgcgcaga tccgcgtatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     6181 gcatcaggaa attgtaagcg ttaatatttt gttaaaattc gattttacca catttgtaga
     6241 ggttttactt gcttaaaaaa acctcccaca tctccccctg aacctgaaac ataaaatgaa
     6301 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     6361 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     6421 actcatcaat gtatcttatc atgtctgctc gaagcggccg ccccaagggg ttatgctagt
     6481 tattgctcag cggtggcagc agccaactca gcttcctttc gggctttgtt agcagccgga
     6541 tcagcttgca tgcctgcagg tcgactctag aggatccccg ggtaccgagc ccgaattcgt
     6601 ttaaacgaag acaggcagcc tcggggacgt ggtcagcgtg caagcattga tatcctcagt
     6661 gtgggctgcc cgatgcaagg atggctcgga agcgctccgg ttgatcttcg gtagagagtg
     6721 ttggagcagc tcaatggaag acaggatctg gggaaaaaga ggcctctctt cctttacttt
     6781 cttcacacag tcagctacca gcctcttcat tgctttgggg cagttcttat atagcttact
     6841 aagatctggg gaggcatatc ctcggcccac catgaagatg atctgatctc ggttgttgat
     6901 gtgagaataa ggaagctccc ccgtcatcag ttcatacaat acgatgccat aggagtagac
     6961 atccgactgg aaactgaatg ggttgttatc ctgcattcgg atcacctctg gggccatcca
     7021 gaggacagag ccagtaggtt gttcaacctg ctgagaacca ctccagcgtg actttactgt
     7081 tgccaaacca aaatctccaa ttttcactgt taagccttca
//
