LOCUS       sequence-375313-        6355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual expression vector for bacteria and mammalian cells producing 
            mScarlet-I-SYFP2 fusion protein for FRET analysis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6355)
  AUTHORS   Gadella TWJ Jr, van Weeren L, Stouthamer J, Hink MA, Wolters AHG, 
            Giepmans BNG, Aumonier S, Dupuy J, Royant A
  TITLE     mScarlet3: a brilliant and fast-maturing red fluorescent protein.
  JOURNAL   Nat Methods. 2023 Apr;20(4):541-545. doi: 
            10.1038/s41592-023-01809-y. Epub 2023 Mar 27.
  PUBMED    36973546
REFERENCE   2  (bases 1 to 6355)
  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: 
            "10.1038/s41592-023-01809-y"; journalName: "Nat Methods"; date: 
            "2023-04"; volume: "20"; issue: "4"; pages: "541-545"
COMMENT     Sequence Label: pDx_mScarlet-I-SYFP2
FEATURES             Location/Qualifiers
     source          1..6355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..37
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     99..118
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        160..278
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             353..370
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             380..397
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      416..425
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             422..1117
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(555..573)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     823..842
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             join(1118..1120,1121..1123,1124..1834)
                     /codon_start=1
                     /product="monomeric optimized variant of EYFP, identical to
                     mVenus but with L68V (Kremers et al., 2006)"
                     /label=SYFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1163..1184)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1424..1443)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1771..1792
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1924..1943)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1989..2044
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2043..2062)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      2132..2179
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     2192..2897
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           2280..2652
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(2309..2897)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(2906..2939)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(3107..3694)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3186..3205)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3707..4522)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4430..4449
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_recomb     4765..5592
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             4765..4889
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(4927..5532)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        5660..5963
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5965..6167
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6118..6138
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa
       61 ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct
      121 gtctcatcat tttggcaaag aattggatcg gaccgaaatc accacaattc agcaaattgt
      181 gaacatcatc acgttcatct ttccctggtt gccaaaggcc cattttcctg tcagtaacga
      241 gaaggtcgcg aattcaggcg ctttttagac tggtcgtagg gagaccacaa cggtttccct
      301 ctagaaataa ttttgtttaa ctataagaag gagatatacc atgcggggtt ctcatcatca
      361 ccaccatcac agcagcggcc tggtgccgcg cggcagctcg ctagcgctac cggtcgccac
      421 catggtgagc aagggcgagg cagtgatcaa ggagttcatg cggttcaagg tgcacatgga
      481 gggctccatg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
      541 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tctcctggga
      601 catcctgtcc cctcagttca tgtacggctc cagggccttc atcaagcacc ccgccgacat
      661 ccccgactac tataagcagt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
      721 cgaggacggc ggcgccgtga ccgtgaccca ggacacctcc ctggaggacg gcaccctgat
      781 ctacaaggtg aagctccgcg gcaccaactt ccctcctgac ggccccgtaa tgcagaagaa
      841 gacaatgggc tgggaagcgt ccaccgagcg gttgtacccc gaggacggcg tgctgaaggg
      901 cgacattaag atggccctgc gcctgaagga cggcggccgc tacctggcgg acttcaagac
      961 cacctacaag gccaagaagc ccgtgcagat gcccggcgcc tacaacgtcg accgcaagtt
     1021 ggacatcacc tcccacaacg aggactacac cgtggtggaa cagtacgaac gctccgaggg
     1081 ccgccactcc accggcggca tggacgagct gtacaagatg gtgagcaagg gcgaggagct
     1141 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1201 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagctgat
     1261 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgggctacgg
     1321 cgtgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1381 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1441 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1501 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     1561 ccacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggcca acttcaagat
     1621 ccgccacaac atcgaggacg gcggcgtgca gctcgccgac cactaccagc agaacacccc
     1681 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc agtccaagct
     1741 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1801 cgggatcact ctcggcatgg acgagctcta caagtaagtg attaacctca ggtgcaggct
     1861 gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata ccactgagat
     1921 cgatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac
     1981 ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct
     2041 ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt
     2101 ttagagtttg gcaacatatg cccatatgta actagcataa ccccttgggg cctctaaacg
     2161 ggtcttgagg ggttttttgc tgaaagcatg cggaggaaat tctccttgaa gtttccctgg
     2221 tgttcaaagt aaaggagttt gcaccagacg cacctctgtt cactggtccg gcgtattaaa
     2281 acacgataca ttgttattag tacatttatt aagcgctaga ttctgtgcgt tgttgattta
     2341 cagacaattg ttgtacgtat tttaataatt cattaaattt ataatcttta gggtggtatg
     2401 ttagagcgaa aatcaaatga ttttcagcgt ctttatatct gaatttaaat attaaatcct
     2461 caatagattt gtaaaatagg tttcgattag tttcaaacaa gggttgtttt tccgaaccga
     2521 tggctggact atctaatgga ttttcgctca acgccacaaa acttgccaaa tcttgtagca
     2581 gcaatctagc tttgtcgata ttcgtttgtg ttttgttttg taataaaggt tcgacgtcgt
     2641 tcaaaatatt atgcgctttt gtatttcttt catcactgtc gttagtatac aattgactcg
     2701 acgtaaacac gttaaataga gcttggacat atttaacatc gggcgtgtta gctttattag
     2761 gccgattatc gtcgtcgtcc caaccctcgt cgttagaagt tgcttccgaa gacgattttg
     2821 ccatagccac acgacgccta ttaattgtgt cggctaacac gtccgcgatc aaatttgtag
     2881 ttgagctttt tggaattgcg atcgcataac ttcgtatagc atacattata cgaagttata
     2941 agctcggaac gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3001 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3061 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3121 gccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3181 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3241 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3301 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3361 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3421 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3481 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3541 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3601 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3661 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgttag aaaaactcat
     3721 cgagcatcaa gtgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     3781 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     3841 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     3901 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     3961 atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     4021 catcaaaatc actcgcacca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     4081 gaaatacgcg atcgccgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     4141 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     4201 ggaatgctgt tttccctggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     4261 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagc ctgaccatct
     4321 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     4381 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     4441 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc gagcaagacg
     4501 tttcccgttg aatatggctc atactcttcc tttttcaata ttattgaagc atttatcagg
     4561 gttattgtct catgtccgcg cgtttcctgc atcttttaat caaatcccaa gatgtgtata
     4621 aacgcgccgg tatgtatagg aagaggttta tactaaactg ttacattgca aacgtggttt
     4681 cgtgtgccaa gtgtgaaaac cgatgtttaa tcaaggctct gacgcatttc tacaaccacg
     4741 actccaagtg tgtgggtgaa gtcatgcatc ttttaatcaa atcccaagat gtgtataaac
     4801 caccaaactg ccaaaaaatg aaaactgtcg acaagctctg tccgtttgct ggcaactgca
     4861 agggtctcaa tcctatttgt aattattgaa taataaaaca attataaatg tcaaatttgt
     4921 tttttattaa cgatacaaac caaacgcaac aagaacattt gtagtattat ctataattga
     4981 aaacgcgtag ttataatcgc tgaggtaata tttaaaatca ttttcaaatg attcacagtt
     5041 aatttgcgac aatataattt tattttcaca taaactagac gccttgtcgt cttcttcttc
     5101 gtattccttc tctttttcat ttttctcttc ataaaaatta acatagttat tatcgtatcc
     5161 atatatgtat ctatcgtata gagtaaattt tttgttgtca taaatatata tgtctttttt
     5221 aatggggtgt atagtaccgc tgcgcatagt ttttctgtaa tttacaacag tgctattttc
     5281 tggtagttct tcggagtgtg ttgctttaat tattaaattt atataatcaa tgaatttggg
     5341 atcgtcggtt ttatacaata tgttgccggc atagtacgca gcttcttcta gttcaattac
     5401 accatttttt agcagcaccg gattaacata actttccaaa atgttgtacg aaccgttaaa
     5461 caaaaacagt tcacctccct tttctatact attgtctgcg agcagttgtt tgttgttaaa
     5521 aataacagcc attgtaatga gacgcacaaa ctaatatcac aaactggaaa tgtctatcaa
     5581 tatatagttg ctgatggccg gcctattaat agtaatcaat tacggggtca ttagttcata
     5641 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     5701 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     5761 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     5821 atcaagtgta tcatatgcca agtccgcccc ctattgacgt caatgacggt aaatggcccg
     5881 cctggcatta tgcccagtac atgaccttac gggactttcc tacttggcag tacatctacg
     5941 tattagtcat cgctattacc atgctgatgc ggttttggca gtacaccaat gggcgtggat
     6001 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     6061 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc ccgttgacgc
     6121 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagacgtcgt ttagtgaacc
     6181 gtcagatcac tagatgcttt attgcggtag tttatcacag ttaaattgct aacgccagtc
     6241 tcgaacttaa cgtgcagaag ttggtcgtga ggcactgggc aggtaagtat cgggcccttt
     6301 gtgcgggggg agcggctcgg ggctgtccgc ggggggacgg ctgccttcgg ggggg
//
