LOCUS       sequence-406385-        8785 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of 2 fluorescently labelled proteins in mammalian cells. 
            Can be used as a CRE-donor (MultiBac system).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8785)
  AUTHORS   Aulicino F, Pelosse M, Toelzer C, Capin J, Ilegems E, Meysami P, 
            Rollarson R, Berggren PO, Dillingham MS, Schaffitzel C, Saleem MA, 
            Welsh GI, Berger I
  TITLE     Highly efficient CRISPR-mediated large DNA docking and multiplexed 
            prime editing using a single baculovirus.
  JOURNAL   Nucleic Acids Res. 2022 Jul 22;50(13):7783-7799. doi: 
            10.1093/nar/gkac587.
  PUBMED    35801912
REFERENCE   2  (bases 1 to 8785)
  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"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2022-07-22- 22"; volume: "50"; issue: 
            "13"; pages: "7783-7799"
COMMENT     Sequence Label: pMMDS 2 colors
FEATURES             Location/Qualifiers
     source          1..8785
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..353
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        354..557
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      629..638
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             635..1312
                     /codon_start=1
                     /product="humanized monomeric Azami-Green fluorescent 
                     protein"
                     /label=hmAzami-Green
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLL
                     LEGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             635..1312
                     /codon_start=1
                     /product="monomeric Azami-Green fluorescent protein 
                     (Karasawa et al., 2003)"
                     /label=mAzami-Green
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGTNFPPNGPVMQKKTLKWEPSTEKMYVEDGVLKGDVNMRLL
                     LEGGGHYRCDFKTTYKAKKEVRLPDAHKIDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     CDS             1355..1390
                     /codon_start=1
                     /product="nanobody epitope tag corresponding to amino acids
                     16-27 of human beta-catenin (Traenkle et al., 2015)"
                     /label=BC2 tag
                     /translation="PDRKAAVSHWQQ"
     polyA_signal    3676..3797
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    3816..3836
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    3864..3884
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    3911..3931
                     /gene="mutant version of attB"
                     /label=attB5
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        3943..4322
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4323..4526
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             4816..5511
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     polyA_signal    5541..5675
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    5796..6384
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5849..5869)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6267..6278)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(6400..6424)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    6523..6556
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      6558..6914
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             7379..8170
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        8261..8370
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     primer_bind     complement(8462..8481)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    8506..8554
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    join(8778..8785,1..17)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 gtacaaaaaa gcaggctgtt taaacgatat ccccatatat ggagttccgc gttacataac
       61 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      121 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      181 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      241 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      301 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      361 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      421 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      481 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      541 tctatataag cagagctgga attctgcagt cgatcgacgg taccgcgggc ccgggccact
      601 agtaacggcc gccagtgtgc tggaattcgc caccatggtg agcgtgatca agcccgagat
      661 gaagatcaag ctgtgcatga ggggcaccgt gaacggccac aacttcgtga tcgagggcga
      721 gggcaagggc aacccctacg agggcaccca gatcctggac ctgaacgtga ccgagggcgc
      781 ccccctgccc ttcgcctacg acatcctgac caccgtgttc cagtacggca acagggcctt
      841 caccaagtac cccgccgaca tccaggacta cttcaagcag accttccccg agggctacca
      901 ctgggagagg agcatgacct acgaggacca gggcatctgc accgccacca gcaacatcag
      961 catgaggggc gactgcttct tctacgacat caggttcgac ggcaccaact tcccccccaa
     1021 cggccccgtg atgcagaaga agaccctgaa gtgggagccc agcaccgaga agatgtacgt
     1081 ggaggacggc gtgctgaagg gcgacgtgaa catgaggctg ctgctggagg gcggcggcca
     1141 ctacaggtgc gacttcaaga ccacctacaa ggccaagaag gaggtgaggc tgcccgacgc
     1201 ccacaagatc gaccacagga tcgagatcct gaagcacgac aaggactaca acaaggtgaa
     1261 gctgtacgag aacgccgtgg ccaggtactc catgctgccc agccaggcca aggctactca
     1321 agctgacctg atggagttgg acatggccat ggagccggac agaaaagctg ctgtcagcca
     1381 ctggcagcag cagtcttact tggattctgg aatccattct ggtgccacca ccacagctcc
     1441 ttccctgagt ggcaagggca accctgagga agaagatgtt gacacctccc aagtccttta
     1501 tgaatgggag caaggctttt cccagtcctt cacgcaagag caagtagctg atattgacgg
     1561 gcagtatgca atgactaggg ctcagagggt ccgagctgcc atgttccctg agacgctaga
     1621 tgagggcatg cagatcccat ccacgcagtt tgacgctgct catcccacta atgtccagcg
     1681 cttggctgaa ccatcacaga tgttgaaaca tgcagttgtc aatttgatta actatcagga
     1741 tgacgcggaa cttgccacac gtgcaattcc tgagctgaca aaactgctaa acgatgagga
     1801 ccaggtggta gttaataaag ctgctgttat ggtccatcag ctttccaaaa aggaagcttc
     1861 cagacatgcc atcatgcgct cccctcagat ggtgtctgcc attgtacgca ccatgcagaa
     1921 tacaaatgat gtagagacag ctcgttgtac tgctgggact ctgcacaacc tttctcacca
     1981 ccgcgagggc ttgctggcca tctttaagtc tggtggcatc ccagcgctgg tgaaaatgct
     2041 tgggtcacca gtggattctg tactgttcta cgccatcacg acactgcata atctcctgct
     2101 ccatcaggaa ggagctaaaa tggcagtgcg cctagctggt ggactgcaga aaatggttgc
     2161 tttgctcaac aaaacaaacg tgaaattctt ggctattaca acagactgcc ttcagatctt
     2221 agcttatggc aatcaagaga gcaagctcat cattctggcc agtggtggac cccaagcctt
     2281 agtaaacata atgaggacct acacttatga gaagcttctg tggaccacaa gcagagtgct
     2341 gaaggtgctg tctgtctgct ctagcaacaa gccggccatt gtagaagctg gtgggatgca
     2401 ggcactgggg cttcatctga cagacccaag tcagcgactt gttcaaaact gtctttggac
     2461 tctcagaaac ctttcagatg cagcgactaa gcaggaaggg atggaaggcc tccttgggac
     2521 tctagtgcag cttctgggtt ccgatgatat aaatgtggtc acctgtgcag ctggaattct
     2581 ctctaacctc acttgcaata attacaaaaa caagatgatg gtgtgccaag tgggtggcat
     2641 agaggctctt gtacgcaccg tccttcgtgc tggtgacagg gaagacatca ctgagcctgc
     2701 catctgtgct cttcgtcatc tgaccagccg gcatcaggaa gccgagatgg cccagaatgc
     2761 cgttcgcctt cattatggac tgcctgttgt ggttaaactc ctgcacccac catcccactg
     2821 gcctctgata aaggcaactg ttggattgat tcgaaacctt gccctttgcc cagcaaatca
     2881 tgcgcctttg cgggaacagg gtgctattcc acgactagtt cagctgcttg tacgagcaca
     2941 tcaggacacc caacggcgca cctccatggg tggaacgcag cagcagtttg tggagggcgt
     3001 gcgcatggag gagatagtag aagggtgtac tggagctctc cacatccttg ctcgggacgt
     3061 tcacaaccgg attgtaatcc gaggactcaa taccattcca ttgtttgtgc agttgcttta
     3121 ttctcccatt gaaaatatcc aaagagtagc tgcaggggtc ctctgtgaac ttgctcagga
     3181 caaggaggct gcagaggcca ttgaagctga gggagccaca gctcccctga cagagttact
     3241 ccactccagg aatgaaggcg tggcaacata cgcagctgct gtcctattcc gaatgtctga
     3301 ggacaagcca caggattaca agaagcggct ttcagtcgag ctgaccagtt ccctcttcag
     3361 gacagagcca atggcttgga atgagactgc agatcttgga ctggacattg gtgcccaggg
     3421 agaagccctt ggatatcgcc aggatgatcc cagctaccgt tcttttcact ctggtggata
     3481 cggccaggat gccttgggga tggaccctat gatggagcat gagatgggtg gccaccaccc
     3541 tggtgctgac tatccagttg atgggctgcc tgatctggga cacgcccagg acctcatgga
     3601 tgggctgccc ccaggtgata gcaatcagct ggcctggttt gatactgacc tgtaatgaat
     3661 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3721 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3781 ctcatcaatg tatcttagat atccctaggc ccgggcaact ttgtataata aagttggttt
     3841 aaacgatatc cctaggcccg ggccaacttt gtatagaaaa gttggtttaa acgatatccc
     3901 taggcccggg caactttgta tacaaaagtt ggtttaaacg atgacattga ttattgacta
     3961 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     4021 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     4081 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     4141 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     4201 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     4261 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     4321 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     4381 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     4441 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     4501 ggtgggaggt ctatataagc agagctgaag tgccattccg cctgacctat gaggcttcgt
     4561 gaacccttgt tgtcaggctc tgctgccatg cccggtgcca gcctgcaaag ggcatgtcgt
     4621 cttctggttg cggtgtgtgc attgcatctc ggggtgactc ttgtgtatta tcttgcggga
     4681 agagatctct cccggcttcc acagttggtg ggcgtgagta cccccttgca aggcggttca
     4741 aatagcgctg cagctattgg acaaagcagc ggcgaactga ggacaggcgg cgctaaagac
     4801 ccaccagtag cgactagcga gctgattaag gagaacatgc acatgaagct gtacatggag
     4861 ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag
     4921 ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac
     4981 atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac ccagggcatc
     5041 cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac
     5101 gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc
     5161 tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat gcagaagaaa
     5221 acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg cctggaaggc
     5281 agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa catcaagacc
     5341 acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac
     5401 tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca gcacgaggtg
     5461 gcagtggcca gatactgcga cctccctagc aaactggggc acaagcttaa tggatcctag
     5521 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     5581 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5641 ccaaactcat caatgtatct tatcatgtct ggatctgatc actgcttgag cctagaagat
     5701 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     5761 ctatcataac ccctagggta tacccatcta attggaatca acctctggat tacaaaattt
     5821 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     5881 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     5941 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     6001 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     6061 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     6121 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     6181 tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc
     6241 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     6301 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     6361 tctccctttg ggccgcctcc ccgcatccct aggcccggga gacagctttc ttgtacaaag
     6421 tggttgataa gcttaaacgt cgagggatct tcataagaga agagggacag ctatgactgg
     6481 gagtagtcag gagaggagga aaaaaaccca tgtgcctggc agataacttc gtataatgta
     6541 tgctatacga agttatggta cgtactaagc tctcatgttt cacgtactaa gctctcatgt
     6601 ttaacgtact aagctctcat gtttaacgaa ctaaaccctc atggctaacg tactaagctc
     6661 tcatggctaa cgtactaagc tctcatgttt cacgtactaa gctctcatgt ttgaacaata
     6721 aaattaatat aaatcagcaa cttaaatagc ctctaaggtt ttaagtttta taagaaaaaa
     6781 aagaatatat aaggctttta aagcttttaa ggtttaacgg ttgtggacaa caagccaggg
     6841 atgtaacgca ctgagaagcc cttagagcct ctcaaagcaa ttttgagtga cacaggaaca
     6901 cttaacggct gacatgggaa ttagcttcac gctgccgcaa gcactcaggg cgcaagggct
     6961 gctaaaggaa gcggaacacg tagaaagcca gtccgcagaa acggtgctga ccccggatga
     7021 atgtcagctg ggaggcagaa taaatgatca tatcgtcaat tattacctcc acggggagag
     7081 cctgagcaaa ctggcctcag gcatttgaga agcacacggt cacactgctt ccggtagtca
     7141 ataaaccggt aagtagcgta tgcgctcacg caactggtcc agaaccttga ccgaacgcag
     7201 cggtggtaac ggcgcagtgg cggttttcat ggcttgttat gactgttttt ttggggtaca
     7261 gtctatgcct cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt
     7321 atggagcagc aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt taaacatcat
     7381 gagggaagcg gtgatcgccg aagtatcgac tcaactatca gaggtagttg gcgtcatcga
     7441 gcgccatctc gaaccgacgt tgctggccgt acatttgtac ggctccgcag tggatggcgg
     7501 cctgaagcca cacagtgata ttgatttgct ggttacggtg accgtaaggc ttgatgaaac
     7561 aacgcggcga gctttgatca acgacctttt ggaaacttcg gcttcccctg gagagagcga
     7621 gattctccgc gctgtagaag tcaccattgt tgtgcacgac gacatcattc cgtggcgtta
     7681 tccagctaag cgcgaactgc aatttggaga atggcagcgc aatgacattc ttgcaggtat
     7741 cttcgagcca gccacgatcg acattgatct ggctatcttg ctgacaaaag caagagaaca
     7801 tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt gatccggttc ctgaacagga
     7861 tctatttgag gcgctaaatg aaaccttaac gctatggaac tcgccgcccg actgggctgg
     7921 cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa
     7981 aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg cccagtatca
     8041 gcccgtcata cttgaagcta gacaggctta tcttggacaa gaagaagatc gcttggcctc
     8101 gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt
     8161 cggcaaataa tgtctaacaa ttcgttcaag ccgacggatc tatgtcgggt gcggagaaag
     8221 aggtaatgaa atggcaccta ggtatcgata ctagtatacg gacctttaat tcaacccaac
     8281 acaatatatt atagttaaat aagaattatt atcaaatcat ttgtatatta attaaaatac
     8341 tatactgtaa attacatttt atttacaatc actcgacgaa gacttgatca cccgggatct
     8401 cgagccatgg tgctagcagc tgatgcatag catgcggtac cgggagatgg gggaggctaa
     8461 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     8521 agaataaaac gcacgggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     8581 tggcactctg tcgatacccc accgagaccc cattgggacc aatacgcccg cgtttcttcc
     8641 ttttccccac cccaaccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     8701 gcggcaagcc ctgccatagc cactacgggt acgtttctgt aactataacg gtcctaaggt
     8761 agcgagttta aacatcaaca agttt
//
