LOCUS       sequence-376972-        6765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fusion of hfYFP and mScarlet-I separated by a TEV cleavage site for 
            fluorescence-assisted protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6765)
  AUTHORS   Campbell BC, Paez-Segala MG, Looger LL, Petsko GA, Liu CF
  TITLE     Chemically stable fluorescent proteins for advanced microscopy.
  JOURNAL   Nat Methods. 2022 Nov 7. pii: 10.1038/s41592-022-01660-7. doi: 
            10.1038/s41592-022-01660-7.
  PUBMED    36344833
REFERENCE   2  (bases 1 to 6765)
  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"; journalName: "Nat 
            Methods. 2022 Nov 7. pii: 10.1038/s41592-022-01660-7. doi: 
            10.1038/s41592-022-01660-7."
COMMENT     Sequence Label: pET28a-hfYFP-TEV-mScarlet-i
FEATURES             Location/Qualifiers
     source          1..6765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      17..472
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(104..123)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     314..335
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(564..1379)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(564..1379)
                     /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
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1287..1306
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1371..1390
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1501..2089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1990..2009
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2243..2260
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2275..2414
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2340..2359)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2500..2522
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2516..2707)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3516..4598)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4560..4579
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4599..4676)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4882..4901
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4985..5004
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4985..5003
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5004..5028
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             5043..5065
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5084..5101
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5180..5201)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             5888..5908
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5912..6604
                     /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="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(6042..6060)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6310..6329
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             6614..6631
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6684..6702)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6698..6745
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
       61 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
      121 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
      181 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
      241 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
      301 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
      361 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
      421 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
      481 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
      541 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
      601 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
      661 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
      721 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
      781 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
      841 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
      901 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
      961 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     1021 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     1081 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     1141 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     1201 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     1261 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     1321 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     1381 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     1441 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     1501 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     1561 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     1621 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     1681 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1741 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1801 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     1861 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     1921 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     1981 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2041 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2101 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     2161 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     2221 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     2281 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     2341 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     2401 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2461 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     2521 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     2581 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     2641 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     2701 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     2761 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     2821 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     2881 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     2941 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     3001 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     3061 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     3121 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     3181 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     3241 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     3301 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     3361 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     3421 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     3481 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3541 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3601 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     3661 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     3721 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     3781 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     3841 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     3901 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     3961 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     4021 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     4081 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     4141 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     4201 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     4261 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     4321 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     4381 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     4441 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     4501 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     4561 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     4621 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     4681 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     4741 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     4801 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     4861 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     4921 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     4981 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     5041 attttgttta actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca
     5101 cagttcaggc aaagcagaaa caatactctc tcatatggtg agcaagggcg aggagctgtt
     5161 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     5221 cgtccgcggc gagggcgagg gcgatgccac caacggcaag ctgaccctga agctcatctc
     5281 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccttag gctacggcct
     5341 gatggtgttc gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     5401 gcccgaaggc tacgtccagg agcgcaccat ctctttcgag gacgacggtt actacaagac
     5461 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgtgc tgaagggcat
     5521 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact tcaacagcca
     5581 caacgtctat atcacggccg acaagcagaa gaacggcatc aaggctaact tcaagatccg
     5641 ccacaacgtt gaggacggcg gcgtgcagct cgccgaccac taccagcaga acacccccat
     5701 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccgtcctgag
     5761 caaagacccc aacgagaagc gcgatcacat ggtcctgaag gagagggtga ccgccgccgg
     5821 gattacacat gacatgaacg agctgtacaa gggatccgct ggctcagctg ctggttctgg
     5881 tgaattcgag aatctgtatt ttcagggtca tgtgagcaag ggcgaggcag tgatcaagga
     5941 gttcatgcgg ttcaaggtgc acatggaggg ctccatgaac ggccacgagt tcgagatcga
     6001 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     6061 gggtggcccc ctgcccttct cctgggacat cctgtcccct cagttcatgt acggctccag
     6121 ggccttcatc aagcaccccg ccgacatccc cgactactat aagcagtcct tccccgaggg
     6181 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gccgtgaccg tgacccagga
     6241 cacctccctg gaggacggca ccctgatcta caaggtgaag ctccgcggca ccaacttccc
     6301 tcctgacggc cccgtaatgc agaagaagac aatgggctgg gaagcgtcca ccgagcggtt
     6361 gtaccccgag gacggcgtgc tgaagggcga cattaagatg gccctgcgcc tgaaggacgg
     6421 cggccgctac ctggcggact tcaagaccac ctacaaggcc aagaagcccg tgcagatgcc
     6481 cggcgcctac aacgtcgacc gcaagttgga catcacctcc cacaacgagg actacaccgt
     6541 ggtggaacag tacgaacgct ccgagggccg ccactccacc ggcggcatgg acgagctgta
     6601 caagtaactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     6661 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     6721 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatc
//
