LOCUS       sequence-385774-        6556 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produces an mTurquoise-antiFRET-mScarlet-I3 fusion protein in 
            bacteria and in mamallian cells it produces 1:1 unfused mTurquoise2 
            and mScarlet-I3 FPs for ratiometric analysis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6556)
  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 6556)
  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: pDRESS_mTurquoise2_spatial-linker-P2A_mScarlet-I3
FEATURES             Location/Qualifiers
     source          1..6556
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     25..852
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             25..149
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(187..792)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        920..1223
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1225..1427
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1378..1398
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1630..1652
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1714..1733
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1775..1893
                     /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             1968..1985
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1995..2012
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2031..2040
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2037..2039,2040..2042,2043..2753)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2082..2103)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2343..2362)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2690..2711
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2874..2930
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     regulatory      2949..2958
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2955..3641
                     /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(3085..3103)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(3740..3759)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3805..3860
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3859..3878)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3948..3995
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4008..4713
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4096..4468
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4125..4713)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4722..4755)
                     /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(4923..5510)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5002..5021)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5523..6338)
                     /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     6246..6265
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 actccaagtg tgtgggtgaa gtcatgcatc ttttaatcaa atcccaagat gtgtataaac
       61 caccaaactg ccaaaaaatg aaaactgtcg acaagctctg tccgtttgct ggcaactgca
      121 agggtctcaa tcctatttgt aattattgaa taataaaaca attataaatg tcaaatttgt
      181 tttttattaa cgatacaaac caaacgcaac aagaacattt gtagtattat ctataattga
      241 aaacgcgtag ttataatcgc tgaggtaata tttaaaatca ttttcaaatg attcacagtt
      301 aatttgcgac aatataattt tattttcaca taaactagac gccttgtcgt cttcttcttc
      361 gtattccttc tctttttcat ttttctcttc ataaaaatta acatagttat tatcgtatcc
      421 atatatgtat ctatcgtata gagtaaattt tttgttgtca taaatatata tgtctttttt
      481 aatggggtgt atagtaccgc tgcgcatagt ttttctgtaa tttacaacag tgctattttc
      541 tggtagttct tcggagtgtg ttgctttaat tattaaattt atataatcaa tgaatttggg
      601 atcgtcggtt ttatacaata tgttgccggc atagtacgca gcttcttcta gttcaattac
      661 accatttttt agcagcaccg gattaacata actttccaaa atgttgtacg aaccgttaaa
      721 caaaaacagt tcacctccct tttctatact attgtctgcg agcagttgtt tgttgttaaa
      781 aataacagcc attgtaatga gacgcacaaa ctaatatcac aaactggaaa tgtctatcaa
      841 tatatagttg ctgatggccg gcctattaat agtaatcaat tacggggtca ttagttcata
      901 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      961 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     1021 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     1081 atcaagtgta tcatatgcca agtccgcccc ctattgacgt caatgacggt aaatggcccg
     1141 cctggcatta tgcccagtac atgaccttac gggactttcc tacttggcag tacatctacg
     1201 tattagtcat cgctattacc atgctgatgc ggttttggca gtacaccaat gggcgtggat
     1261 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     1321 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc ccgttgacgc
     1381 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagacgtcgt ttagtgaacc
     1441 gtcagatcac tagatgcttt attgcggtag tttatcacag ttaaattgct aacgccagtc
     1501 tcgaacttaa cgtgcagaag ttggtcgtga ggcactgggc aggtaagtat cgggcccttt
     1561 gtgcgggggg agcggctcgg ggctgtccgc ggggggacgg ctgccttcgg gggggacggg
     1621 gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct gctaaccatg
     1681 ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt gtgctgtctc
     1741 atcattttgg caaagaattg gatcggaccg aaatcaccac aattcagcaa attgtgaaca
     1801 tcatcacgtt catctttccc tggttgccaa aggcccattt tcctgtcagt aacgagaagg
     1861 tcgcgaattc aggcgctttt tagactggtc gtagggagac cacaacggtt tccctctaga
     1921 aataattttg tttaactata agaaggagat ataccatgcg gggttctcat catcaccacc
     1981 atcacagcag cggcctggtg ccgcgcggca gctcgctagc gctgccggtc gccaccatgg
     2041 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2101 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2161 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2221 tgaccaccct gtcctggggc gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc
     2281 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2341 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2401 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2461 tggagtacaa ctactttagc gacaacgtct atatcaccgc cgacaagcag aagaacggca
     2521 tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc
     2581 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2641 tgagcaccca gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2701 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctctac aagtcgacca
     2761 gatctctggc cgaggcggca gctaaggagg ccgcggcgaa ggaggcagcc gcgaaagaag
     2821 cagccgcaaa ggaagcggcc gccaaggctg ctgcaagatc tctcggatcg ggtgctacta
     2881 acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct tcgctagcgc
     2941 taccggtcgc caccatggat agcaccgagg cagtgatcaa ggagttcatg cggttcaagg
     3001 tgcacatgga gggctccatg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     3061 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct
     3121 tctcctggga catcctgtcc cctcagttca tgtacggctc cagggccttc atcaagcacc
     3181 ccgccgacat ccccgactac tggaagcagt ccttccccga gggcttcaag tgggagcgcg
     3241 tgatgatctt cgaggacggc ggcaccgtgt ccgtgaccca ggacacctcc ctggaggacg
     3301 gcaccctgat ctacaaggtg aagctccgcg gcggcaactt ccctcctgac ggccccgtaa
     3361 tgcagaagag gacaatgggc tgggaagcat ccaccgagcg gttgtacccc gaggacgtcg
     3421 tgctgaaggg cgacattaag atggccctgc gcctgaagga cggcggccgc tacctggcgg
     3481 acttcaagac cacctacaag gccaagaagc ccgtgcagat gcccggcgcc ttcaacatcg
     3541 accgcaagtt ggacatcacc tcccacaacg aggactacac cgtggtggaa cagtacgaac
     3601 gctccgtggc ccgccactcc accggcggct ccggtggctc cttgtacaag taagtgatta
     3661 acctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca
     3721 caaataccac tgagatcgat ctttttccct ctgccaaaaa ttatggggac atcatgaagc
     3781 cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt
     3841 ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat ttaaaacatc
     3901 agaatgagta tttggtttag agtttggcaa catatgccca tatgtaacta gcataacccc
     3961 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa agcatgcgga ggaaattctc
     4021 cttgaagttt ccctggtgtt caaagtaaag gagtttgcac cagacgcacc tctgttcact
     4081 ggtccggcgt attaaaacac gatacattgt tattagtaca tttattaagc gctagattct
     4141 gtgcgttgtt gatttacaga caattgttgt acgtatttta ataattcatt aaatttataa
     4201 tctttagggt ggtatgttag agcgaaaatc aaatgatttt cagcgtcttt atatctgaat
     4261 ttaaatatta aatcctcaat agatttgtaa aataggtttc gattagtttc aaacaagggt
     4321 tgtttttccg aaccgatggc tggactatct aatggatttt cgctcaacgc cacaaaactt
     4381 gccaaatctt gtagcagcaa tctagctttg tcgatattcg tttgtgtttt gttttgtaat
     4441 aaaggttcga cgtcgttcaa aatattatgc gcttttgtat ttctttcatc actgtcgtta
     4501 gtatacaatt gactcgacgt aaacacgtta aatagagctt ggacatattt aacatcgggc
     4561 gtgttagctt tattaggccg attatcgtcg tcgtcccaac cctcgtcgtt agaagttgct
     4621 tccgaagacg attttgccat agccacacga cgcctattaa ttgtgtcggc taacacgtcc
     4681 gcgatcaaat ttgtagttga gctttttgga attgcgatcg cataacttcg tatagcatac
     4741 attatacgaa gttataagct cggaacgctg cgctcggtcg ttcggctgcg gcgagcggta
     4801 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4861 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4921 tttttccata ggctccgccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4981 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5041 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5101 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5161 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5221 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5281 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5341 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5401 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5461 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5521 tgttagaaaa actcatcgag catcaagtga aactgcaatt tattcatatc aggattatca
     5581 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     5641 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     5701 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     5761 actgaatccg gtgagaatgg caaaagctta tgcatttctt tccagacttg ttcaacaggc
     5821 cagccattac gctcgtcatc aaaatcactc gcaccaacca aaccgttatt cattcgtgat
     5881 tgcgcctgag cgagacgaaa tacgcgatcg ccgttaaaag gacaattaca aacaggaatc
     5941 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     6001 tattcttcta atacctggaa tgctgttttc cctgggatcg cagtggtgag taaccatgca
     6061 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     6121 tttagcctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     6181 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     6241 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     6301 ggcctcgagc aagacgtttc ccgttgaata tggctcatac tcttcctttt tcaatattat
     6361 tgaagcattt atcagggtta ttgtctcatg tccgcgcgtt tcctgcatct tttaatcaaa
     6421 tcccaagatg tgtataaacg cgccggtatg tataggaaga ggtttatact aaactgttac
     6481 attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat gtttaatcaa ggctctgacg
     6541 catttctaca accacg
//
