LOCUS       sequence-375318-        6355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual expression vector for bacteria and mammalian cells producing 
            mScarlet-I3-NCwt-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-I3-NCwt-SYFP2
FEATURES             Location/Qualifiers
     source          1..6355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..35
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     97..116
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        158..276
                     /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             351..368
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             378..395
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      414..423
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             420..1115
                     /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
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYWKQSFPEGFKWERVMIFEDGG
                     TVSVTQDTSLEDGTLIYKVKLRGGNFPPDGPVMQKRTMGWEASTERLYPEDVVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARH
                     STGGMDELYK"
     primer_bind     complement(553..571)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             join(1116..1118,1119..1121,1122..1832)
                     /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(1161..1182)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1422..1441)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1769..1790
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1922..1941)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1987..2042
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2041..2060)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      2130..2177
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     2190..2895
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           2278..2650
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(2307..2895)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(2904..2937)
                     /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(3105..3692)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3184..3203)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3705..4520)
                     /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     4428..4447
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_recomb     4763..5590
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             4763..4887
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(4925..5530)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        5658..5961
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5963..6165
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6116..6136
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc
       61 atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt
      121 ctcatcattt tggcaaagaa ttggatcgga ccgaaatcac cacaattcag caaattgtga
      181 acatcatcac gttcatcttt ccctggttgc caaaggccca ttttcctgtc agtaacgaga
      241 aggtcgcgaa ttcaggcgct ttttagactg gtcgtaggga gaccacaacg gtttccctct
      301 agaaataatt ttgtttaact ataagaagga gatataccat gcggggttct catcatcacc
      361 accatcacag cagcggcctg gtgccgcgcg gcagctcgct agcgctgccg gtcgccacca
      421 tggtgagcaa gggcgaggca gtgatcaagg agttcatgcg gttcaaggtg cacatggagg
      481 gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
      541 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc tcctgggaca
      601 tcctgtcccc tcagttcatg tacggctcca gggccttcat caagcacccc gccgacatcc
      661 ccgactactg gaagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg atgatcttcg
      721 aggacggcgg caccgtgtcc gtgacccagg acacctccct ggaggacggc accctgatct
      781 acaaggtgaa gctccgcggc ggcaacttcc ctcctgacgg ccccgtaatg cagaagagga
      841 caatgggctg ggaagcatcc accgagcggt tgtaccccga ggacgtcgtg ctgaagggcg
      901 acattaagat ggccctgcgc ctgaaggacg gcggccgcta cctggcggac ttcaagacca
      961 cctacaaggc caagaagccc gtgcagatgc ccggcgcctt caacatcgac cgcaagttgg
     1021 acatcacctc ccacaacgag gactacaccg tggtggaaca gtacgaacgc tccgtggccc
     1081 gccactccac cggcggcatg gacgagctgt acaagatggt gagcaagggc gaggagctgt
     1141 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1201 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagctgatct
     1261 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg ggctacggcg
     1321 tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1381 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     1441 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1501 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     1561 acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggccaac ttcaagatcc
     1621 gccacaacat cgaggacggc ggcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1681 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag tccaagctga
     1741 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     1801 ggatcactct cggcatggac gagctctaca agtaagtgat taacctcagg tgcaggctgc
     1861 ctatcagaag gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatcg
     1921 atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt
     1981 ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct
     2041 cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt
     2101 agagtttggc aacatatgcc catatgtaac tagcataacc ccttggggcc tctaaacggg
     2161 tcttgagggg ttttttgctg aaagcatgcg gaggaaattc tccttgaagt ttccctggtg
     2221 ttcaaagtaa aggagtttgc accagacgca cctctgttca ctggtccggc gtattaaaac
     2281 acgatacatt gttattagta catttattaa gcgctagatt ctgtgcgttg ttgatttaca
     2341 gacaattgtt gtacgtattt taataattca ttaaatttat aatctttagg gtggtatgtt
     2401 agagcgaaaa tcaaatgatt ttcagcgtct ttatatctga atttaaatat taaatcctca
     2461 atagatttgt aaaataggtt tcgattagtt tcaaacaagg gttgtttttc cgaaccgatg
     2521 gctggactat ctaatggatt ttcgctcaac gccacaaaac ttgccaaatc ttgtagcagc
     2581 aatctagctt tgtcgatatt cgtttgtgtt ttgttttgta ataaaggttc gacgtcgttc
     2641 aaaatattat gcgcttttgt atttctttca tcactgtcgt tagtatacaa ttgactcgac
     2701 gtaaacacgt taaatagagc ttggacatat ttaacatcgg gcgtgttagc tttattaggc
     2761 cgattatcgt cgtcgtccca accctcgtcg ttagaagttg cttccgaaga cgattttgcc
     2821 atagccacac gacgcctatt aattgtgtcg gctaacacgt ccgcgatcaa atttgtagtt
     2881 gagctttttg gaattgcgat cgcataactt cgtatagcat acattatacg aagttataag
     2941 ctcggaacgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3001 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3061 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3121 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3181 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3241 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3301 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3361 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3421 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3481 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3541 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3601 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3661 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgttagaa aaactcatcg
     3721 agcatcaagt gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     3781 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     3841 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     3901 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     3961 ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     4021 tcaaaatcac tcgcaccaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     4081 aatacgcgat cgccgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     4141 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     4201 aatgctgttt tccctgggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     4261 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagcct gaccatctca
     4321 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     4381 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     4441 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt
     4501 tcccgttgaa tatggctcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4561 tattgtctca tgtccgcgcg tttcctgcat cttttaatca aatcccaaga tgtgtataaa
     4621 cgcgccggta tgtataggaa gaggtttata ctaaactgtt acattgcaaa cgtggtttcg
     4681 tgtgccaagt gtgaaaaccg atgtttaatc aaggctctga cgcatttcta caaccacgac
     4741 tccaagtgtg tgggtgaagt catgcatctt ttaatcaaat cccaagatgt gtataaacca
     4801 ccaaactgcc aaaaaatgaa aactgtcgac aagctctgtc cgtttgctgg caactgcaag
     4861 ggtctcaatc ctatttgtaa ttattgaata ataaaacaat tataaatgtc aaatttgttt
     4921 tttattaacg atacaaacca aacgcaacaa gaacatttgt agtattatct ataattgaaa
     4981 acgcgtagtt ataatcgctg aggtaatatt taaaatcatt ttcaaatgat tcacagttaa
     5041 tttgcgacaa tataatttta ttttcacata aactagacgc cttgtcgtct tcttcttcgt
     5101 attccttctc tttttcattt ttctcttcat aaaaattaac atagttatta tcgtatccat
     5161 atatgtatct atcgtataga gtaaattttt tgttgtcata aatatatatg tcttttttaa
     5221 tggggtgtat agtaccgctg cgcatagttt ttctgtaatt tacaacagtg ctattttctg
     5281 gtagttcttc ggagtgtgtt gctttaatta ttaaatttat ataatcaatg aatttgggat
     5341 cgtcggtttt atacaatatg ttgccggcat agtacgcagc ttcttctagt tcaattacac
     5401 cattttttag cagcaccgga ttaacataac tttccaaaat gttgtacgaa ccgttaaaca
     5461 aaaacagttc acctcccttt tctatactat tgtctgcgag cagttgtttg ttgttaaaaa
     5521 taacagccat tgtaatgaga cgcacaaact aatatcacaa actggaaatg tctatcaata
     5581 tatagttgct gatggccggc ctattaatag taatcaatta cggggtcatt agttcatagc
     5641 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     5701 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     5761 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     5821 caagtgtatc atatgccaag tccgccccct attgacgtca atgacggtaa atggcccgcc
     5881 tggcattatg cccagtacat gaccttacgg gactttccta cttggcagta catctacgta
     5941 ttagtcatcg ctattaccat gctgatgcgg ttttggcagt acaccaatgg gcgtggatag
     6001 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     6061 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa
     6121 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gacgtcgttt agtgaaccgt
     6181 cagatcacta gatgctttat tgcggtagtt tatcacagtt aaattgctaa cgccagtctc
     6241 gaacttaacg tgcagaagtt ggtcgtgagg cactgggcag gtaagtatcg ggccctttgt
     6301 gcggggggag cggctcgggg ctgtccgcgg ggggacggct gccttcgggg gggac
//
