LOCUS       sequence-376985-        6765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fusion of LSSmGFP 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-LSSmGFP-TEV-mScarlet-i
FEATURES             Location/Qualifiers
     source          1..6765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      26..481
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(113..132)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     323..344
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(573..1388)
                     /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(573..1388)
                     /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     1296..1315
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1380..1399
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1510..2098
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1999..2018
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2252..2269
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2284..2423
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2349..2368)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2509..2531
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2525..2716)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3525..4607)
                     /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     4569..4588
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4608..4685)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4891..4910
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4994..5013
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4994..5012
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5013..5037
                     /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             5052..5074
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5093..5110
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5189..5210)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             5897..5917
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5921..6613
                     /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(6051..6069)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6319..6338
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             6623..6640
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6693..6711)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6707..6754
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
       61 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
      121 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
      181 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
      241 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
      301 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
      361 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
      421 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
      481 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
      541 attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga
      601 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
      661 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
      721 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
      781 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta
      841 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
      901 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
      961 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     1021 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     1081 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     1141 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     1201 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     1261 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     1321 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata
     1381 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac
     1441 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1501 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1561 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1621 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     1681 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1741 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1801 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1861 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1921 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1981 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2041 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     2101 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2161 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     2221 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     2281 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     2341 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     2401 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     2461 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     2521 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     2581 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     2641 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     2701 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     2761 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     2821 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     2881 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     2941 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     3001 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     3061 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     3121 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     3181 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     3241 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     3301 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     3361 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     3421 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     3481 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     3541 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3601 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     3661 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     3721 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     3781 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     3841 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     3901 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     3961 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     4021 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     4081 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     4141 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     4201 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     4261 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     4321 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     4381 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     4441 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     4501 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     4561 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     4621 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     4681 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     4741 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     4801 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     4861 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     4921 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     4981 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     5041 ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca
     5101 tcatcatcac agttcaggca aagcagaaac aatactctct catatggtga gcaagggcga
     5161 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     5221 caagttcagc gtccgcggcg agggcgaggg cgatgccacc aacggcaaac tgtccctgaa
     5281 gctcatctcc accaccggca agctgcccgt gccctggccc accctcgtga ccaccttaag
     5341 ctacggccag atggtgttcg cccgctaccc tgacaacatg aagcagcacg acttcttcaa
     5401 gtccgccatg cccgaaggct acgtccagga gcgcaccatc tctttcgagg acgacggtta
     5461 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgtgct
     5521 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaacctgg agtacaactt
     5581 caacagccac aacgtctata tcacggccga caagcagaag aacggcatca aggctaactt
     5641 caagatccgc cacaacgttg aggacggcgg cgtgcagctc gccgaccact accagcagaa
     5701 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcatccagtc
     5761 caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgaagg agagggtgac
     5821 cgccgccggg attacacatg acatgaacga gctgtacaag ggatccgctg gctcagctgc
     5881 tggttctggt gaattcgaga atctgtattt tcagggtcat gtgagcaagg gcgaggcagt
     5941 gatcaaggag ttcatgcggt tcaaggtgca catggagggc tccatgaacg gccacgagtt
     6001 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     6061 ggtgaccaag ggtggccccc tgcccttctc ctgggacatc ctgtcccctc agttcatgta
     6121 cggctccagg gccttcatca agcaccccgc cgacatcccc gactactata agcagtcctt
     6181 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg ccgtgaccgt
     6241 gacccaggac acctccctgg aggacggcac cctgatctac aaggtgaagc tccgcggcac
     6301 caacttccct cctgacggcc ccgtaatgca gaagaagaca atgggctggg aagcgtccac
     6361 cgagcggttg taccccgagg acggcgtgct gaagggcgac attaagatgg ccctgcgcct
     6421 gaaggacggc ggccgctacc tggcggactt caagaccacc tacaaggcca agaagcccgt
     6481 gcagatgccc ggcgcctaca acgtcgaccg caagttggac atcacctccc acaacgagga
     6541 ctacaccgtg gtggaacagt acgaacgctc cgagggccgc cactccaccg gcggcatgga
     6601 cgagctgtac aagtaactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
     6661 agcccgagag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     6721 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggagg
//
