LOCUS       sequence-375316-        6352 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual expression vector for bacteria and mammalian cells producing 
            mScarlet-I3-SYFP2 fusion protein for FRET analysis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6352)
  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 6352)
  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-I3-SYFP2
FEATURES             Location/Qualifiers
     source          1..6352
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     17..39
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     101..120
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        162..280
                     /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             355..372
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             382..399
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      418..427
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             424..1110
                     /codon_start=1
                     /product="bright cysteine-free monomeric red fluorescent 
                     protein with fast and complete maturation (Gadella et al., 
                     2023)"
                     /label=mScarlet3
                     /translation="MDSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYWKQSFPEGFKWERVMIFEDGGT
                     VSVTQDTSLEDGTLIYKVKLRGGNFPPDGPVMQKRTMGWEASTERLYPEDVVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHS
                     TGGSGGS"
     primer_bind     complement(554..572)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             join(1120..1122,1123..1833)
                     /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="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1162..1183)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1423..1442)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1770..1791
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1923..1942)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1988..2043
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2042..2061)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      2131..2178
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     2191..2896
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           2279..2651
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(2308..2896)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(2905..2938)
                     /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(3106..3693)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3185..3204)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3706..4521)
                     /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     4429..4448
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_recomb     4764..5591
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             4764..4888
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(4926..5531)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        5659..5962
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5964..6166
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6117..6137
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct
       61 aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg
      121 ctgtctcatc attttggcaa agaattggat cggaccgaaa tcaccacaat tcagcaaatt
      181 gtgaacatca tcacgttcat ctttccctgg ttgccaaagg cccattttcc tgtcagtaac
      241 gagaaggtcg cgaattcagg cgctttttag actggtcgta gggagaccac aacggtttcc
      301 ctctagaaat aattttgttt aactataaga aggagatata ccatgcgggg ttctcatcat
      361 caccaccatc acagcagcgg cctggtgccg cgcggcagct cgctagcgct gccggtcgcc
      421 accatggata gcaccgaggc agtgatcaag gagttcatgc ggttcaaggt gcacatggag
      481 ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
      541 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt ctcctgggac
      601 atcctgtccc ctcagttcat gtacggctcc agggccttca tcaagcaccc cgccgacatc
      661 cccgactact ggaagcagtc cttccccgag ggcttcaagt gggagcgcgt gatgatcttc
      721 gaggacggcg gcaccgtgtc cgtgacccag gacacctccc tggaggacgg caccctgatc
      781 tacaaggtga agctccgcgg cggcaacttc cctcctgacg gccccgtaat gcagaagagg
      841 acaatgggct gggaagcatc caccgagcgg ttgtaccccg aggacgtcgt gctgaagggc
      901 gacattaaga tggccctgcg cctgaaggac ggcggccgct acctggcgga cttcaagacc
      961 acctacaagg ccaagaagcc cgtgcagatg cccggcgcct tcaacatcga ccgcaagttg
     1021 gacatcacct cccacaacga ggactacacc gtggtggaac agtacgaacg ctccgtggcc
     1081 cgccactcca ccggcggctc cggtggctcc ttgtacaagg tgagcaaggg cgaggagctg
     1141 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     1201 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagctgatc
     1261 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gggctacggc
     1321 gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     1381 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     1441 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     1501 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     1561 cacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc
     1621 cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc
     1681 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccaagctg
     1741 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1801 gggatcactc tcggcatgga cgagctctac aagtaagtga ttaacctcag gtgcaggctg
     1861 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc
     1921 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     1981 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     2041 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     2101 tagagtttgg caacatatgc ccatatgtaa ctagcataac cccttggggc ctctaaacgg
     2161 gtcttgaggg gttttttgct gaaagcatgc ggaggaaatt ctccttgaag tttccctggt
     2221 gttcaaagta aaggagtttg caccagacgc acctctgttc actggtccgg cgtattaaaa
     2281 cacgatacat tgttattagt acatttatta agcgctagat tctgtgcgtt gttgatttac
     2341 agacaattgt tgtacgtatt ttaataattc attaaattta taatctttag ggtggtatgt
     2401 tagagcgaaa atcaaatgat tttcagcgtc tttatatctg aatttaaata ttaaatcctc
     2461 aatagatttg taaaataggt ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat
     2521 ggctggacta tctaatggat tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag
     2581 caatctagct ttgtcgatat tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt
     2641 caaaatatta tgcgcttttg tatttctttc atcactgtcg ttagtataca attgactcga
     2701 cgtaaacacg ttaaatagag cttggacata tttaacatcg ggcgtgttag ctttattagg
     2761 ccgattatcg tcgtcgtccc aaccctcgtc gttagaagtt gcttccgaag acgattttgc
     2821 catagccaca cgacgcctat taattgtgtc ggctaacacg tccgcgatca aatttgtagt
     2881 tgagcttttt ggaattgcga tcgcataact tcgtatagca tacattatac gaagttataa
     2941 gctcggaacg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3001 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3061 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3121 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3181 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3241 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3301 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3361 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3421 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3481 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3541 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3601 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3661 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgttaga aaaactcatc
     3721 gagcatcaag tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     3781 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     3841 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     3901 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     3961 tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     4021 atcaaaatca ctcgcaccaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     4081 aaatacgcga tcgccgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     4141 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     4201 gaatgctgtt ttccctggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     4261 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagcc tgaccatctc
     4321 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     4381 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     4441 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg agcaagacgt
     4501 ttcccgttga atatggctca tactcttcct ttttcaatat tattgaagca tttatcaggg
     4561 ttattgtctc atgtccgcgc gtttcctgca tcttttaatc aaatcccaag atgtgtataa
     4621 acgcgccggt atgtatagga agaggtttat actaaactgt tacattgcaa acgtggtttc
     4681 gtgtgccaag tgtgaaaacc gatgtttaat caaggctctg acgcatttct acaaccacga
     4741 ctccaagtgt gtgggtgaag tcatgcatct tttaatcaaa tcccaagatg tgtataaacc
     4801 accaaactgc caaaaaatga aaactgtcga caagctctgt ccgtttgctg gcaactgcaa
     4861 gggtctcaat cctatttgta attattgaat aataaaacaa ttataaatgt caaatttgtt
     4921 ttttattaac gatacaaacc aaacgcaaca agaacatttg tagtattatc tataattgaa
     4981 aacgcgtagt tataatcgct gaggtaatat ttaaaatcat tttcaaatga ttcacagtta
     5041 atttgcgaca atataatttt attttcacat aaactagacg ccttgtcgtc ttcttcttcg
     5101 tattccttct ctttttcatt tttctcttca taaaaattaa catagttatt atcgtatcca
     5161 tatatgtatc tatcgtatag agtaaatttt ttgttgtcat aaatatatat gtctttttta
     5221 atggggtgta tagtaccgct gcgcatagtt tttctgtaat ttacaacagt gctattttct
     5281 ggtagttctt cggagtgtgt tgctttaatt attaaattta tataatcaat gaatttggga
     5341 tcgtcggttt tatacaatat gttgccggca tagtacgcag cttcttctag ttcaattaca
     5401 ccatttttta gcagcaccgg attaacataa ctttccaaaa tgttgtacga accgttaaac
     5461 aaaaacagtt cacctccctt ttctatacta ttgtctgcga gcagttgttt gttgttaaaa
     5521 ataacagcca ttgtaatgag acgcacaaac taatatcaca aactggaaat gtctatcaat
     5581 atatagttgc tgatggccgg cctattaata gtaatcaatt acggggtcat tagttcatag
     5641 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     5701 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     5761 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     5821 tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc
     5881 ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt
     5941 attagtcatc gctattacca tgctgatgcg gttttggcag tacaccaatg ggcgtggata
     6001 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     6061 ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca
     6121 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agacgtcgtt tagtgaaccg
     6181 tcagatcact agatgcttta ttgcggtagt ttatcacagt taaattgcta acgccagtct
     6241 cgaacttaac gtgcagaagt tggtcgtgag gcactgggca ggtaagtatc gggccctttg
     6301 tgcgggggga gcggctcggg gctgtccgcg gggggacggc tgccttcggg gg
//
