LOCUS       sequence-479074-        6222 bp    DNA     circular SYN 21-JUL-2025
DEFINITION  Express CRAF-HiBiT 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 6222)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6222)
  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            "Unpublished"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: CRAF-HiBiT Fusion Vector
FEATURES             Location/Qualifiers
     source          1..6222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1033..1052
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1033..1051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3165..3183)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3220..3341
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3231..3250)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3285..3304
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3586..3943
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3586..3777
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3609..3629)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3794..3929
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3958..4005
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4037..4831
                     /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(4091..4110)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4701..4720
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4895..4943
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(5182..5770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5262..5281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    5886..6169
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcgat cgccatggag cacatacagg
     1081 gagcttggaa gacgatcagc aatggttttg gattcaaaga tgccgtgttt gatggctcca
     1141 gctgcatctc tcctacaata gttcagcagt ttggctatca gcgccgggca tcagatgatg
     1201 gcaaactcac agatccttct aagacaagca acactatccg tgttttcttg ccgaacaagc
     1261 aaagaacagt ggtcaatgtg cgaaatggaa tgagcttgca tgactgcctt atgaaagcac
     1321 tcaaggtgag gggcctgcaa ccagagtgct gtgcagtgtt cagacttctc cacgaacaca
     1381 aaggtaaaaa agcacgctta gattggaata ctgatgctgc gtctttgatt ggagaagaac
     1441 ttcaagtaga tttcctggat catgttcccc tcacaacaca caactttgct cggaagacgt
     1501 tcctgaagct tgccttctgt gacatctgtc agaaattcct gctcaatgga tttcgatgtc
     1561 agacttgtgg ctacaaattt catgagcact gtagcaccaa agtacctact atgtgtgtgg
     1621 actggagtaa catcagacaa ctcttattgt ttccaaattc cactattggt gatagtggag
     1681 tcccagcact accttctttg actatgcgtc gtatgcgaga gtctgtttcc aggatgcctg
     1741 ttagttctca gcacagatat tctacacctc acgccttcac ctttaacacc tccagtccct
     1801 catctgaagg ttccctctcc cagaggcaga ggtcgacatc cacacctaat gtccacatgg
     1861 tcagcaccac cctgcctgtg gacagcagga tgattgagga tgcaattcga agtcacagcg
     1921 aatcagcctc accttcagcc ctgtccagta gccccaacaa tctgagccca acaggctggt
     1981 cacagccgaa aacccccgtg ccagcacaaa gagagcgggc accagtatct gggacccagg
     2041 agaaaaacaa aattaggcct cgtggacaga gagattcaag ctattattgg gaaatagaag
     2101 ccagtgaagt gatgctgtcc actcggattg ggtcaggctc ttttggaact gtttataagg
     2161 gtaaatggca cggagatgtt gcagtaaaga tcctaaaggt tgtcgaccca accccagagc
     2221 aattccaggc cttcaggaat gaggtggctg ttctgcgcaa aacacggcat gtgaacattc
     2281 tgcttttcat ggggtacatg acaaaggaca acctggcaat tgtgacccag tggtgcgagg
     2341 gcagcagcct ctacaaacac ctgcatgtcc aggagaccaa gtttcagatg ttccagctaa
     2401 ttgacattgc ccggcagacg gctcagggaa tggactattt gcatgcaaag aacatcatcc
     2461 atagagacat gaaatccaac aatatatttc tccatgaagg cttaacagtg aaaattggag
     2521 attttggttt ggcaacagta aagtcacgct ggagtggttc tcagcaggtt gaacaaccta
     2581 ctggctctgt cctctggatg gccccagagg tgatccgaat gcaggataac aacccattca
     2641 gtttccagtc ggatgtctac tcctatggca tcgtattgta tgaactgatg acgggggagc
     2701 ttccttattc tcacatcaac aaccgagatc agatcatctt catggtgggc cgaggatatg
     2761 cctccccaga tcttagtaag ctatataaga actgccccaa agcaatgaag aggctggtag
     2821 ctgactgtgt gaagaaagta aaggaagaga ggcctctttt tccccagatc ctgtcttcca
     2881 ttgagctgct ccaacactct ctaccgaaga tcaaccggag cgcttccgag ccatccttgc
     2941 atcgggcagc ccacactgag gatatcaatg cttgcacgct gaccacgtcc ccgaggctgc
     3001 ctgtcttcgt ttctcaaggc agttcaggtg gctcgagcgg tgtgagcggc tggcggctgt
     3061 tcaagaagat tagctaaggc cgcgactcta gagtcgacct gcaggcatgc aagctgatcc
     3121 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     3181 agcataaccc cttggggcgg ccgcttcgag cagacatgat aagatacatt gatgagtttg
     3241 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     3301 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     3361 attttatgtt tcaggttcag ggggagatgt gggaggtttt tttaagcaag taaaacctct
     3421 acaaatgtgg taaaatcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt
     3481 attttctcct tacgcatctg tgcggtattt cacaccgcat acgcggatct gcgcagcacc
     3541 atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag gcggaaagaa
     3601 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3661 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     3721 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3781 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3841 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     3901 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctta attaactgtt
     3961 gacaattaat catcggcata gtatgtcggc atagtataat acgacaaggt gaggaactaa
     4021 acccaggagg cagatcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     4081 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4141 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4201 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4261 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4321 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4381 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4441 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4501 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4561 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     4621 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     4681 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     4741 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4801 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     4861 caagcgacgc ccaacctgcc atcacgatgg ccgcaataaa atatctttat tttcattaca
     4921 tctgtgtgtt ggttttttgt gtgaatcgat agcgataagg atcctctttg cgcttgcgtt
     4981 ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc gtttctgcgg
     5041 actggctttc tacccggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5101 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5161 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5221 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5281 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5341 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5401 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5461 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5521 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5581 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     5641 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5701 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5761 aggatttcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5821 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5881 atagtccgga aatacaggaa cgcacgctgg atggcccttc gctgggatgg tgaaaccatg
     5941 aaaaatggca gcttcagtgg attaagtggg ggtaatgtgg cctgtaccct ctggttgcat
     6001 aggtattcat acggttaaaa tttatcaggc gcgattgcgg cagtttttcg ggtggtttgt
     6061 tgccattttt acctgtctgc tgccgtgatc gcgctgaacg cgttttagcg gtgcgtacaa
     6121 ttaagggatt atggtaaatc cacttactgt ctgccctcgt agccatcgag ataaaccgca
     6181 gtactccggc cacgatgcgt ccggcgtaga ggatcgagat ct
//
