LOCUS       sequence-267049-        6024 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyTag003-mKate2 (a far-red fluorescent protein) in 
            bacterial cytoplasm.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6024)
  AUTHORS   Keeble AH, Turkki P, Stokes S, Khairil Anuar INA, Rahikainen R, 
            Hytonen VP, Howarth M
  TITLE     Approaching infinite affinity through engineering of peptide-protein
            interaction.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Dec 10. pii: 1909653116. doi: 
            10.1073/pnas.1909653116.
  PUBMED    31822621
REFERENCE   2  (bases 1 to 6024)
  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: "Proc Natl 
            Acad Sci U S A. 2019 Dec 10. pii: 1909653116. doi: 
            10.1073/pnas.1909653116."
COMMENT     Sequence Label: SpyTag003-mKate2
FEATURES             Location/Qualifiers
     source          1..6024
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             15..37
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             51..98
                     /codon_start=1
                     /product="improved SpyTag variant that reacts with 
                     SpyCatcher003 at a rate approaching the diffusion limit 
                     (Keeble et al., 2019)"
                     /label=SpyTag003
                     /translation="RGVPHIVMVDAYKRYK"
     CDS             117..809
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHR"
     CDS             828..845
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(915..933)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      929..976
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1013..1468
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1100..1119)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1310..1331
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1560..2375)
                     /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(1560..2375)
                     /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     2283..2302
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2367..2386
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2497..3085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2986..3005
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3239..3256
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3271..3410
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3336..3355)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3496..3518
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3512..3703)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4512..5594)
                     /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     5556..5575
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5595..5672)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5878..5897
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5981..6000
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5981..5999
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6000..6024
                     /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)."
ORIGIN
        1 cctctagaaa taattttgtt taactttaag aaggagatat acatatggga cgtggcgttc
       61 ctcatattgt tatggtggac gcctacaaac gctataaatc gggaggtggt tcaggcgtga
      121 gcgagctgat taaggagaac atgcacatga agctgtacat ggagggcacc gtgaacaacc
      181 accacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga
      241 gaatcaaggc cgtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctaccagct
      301 tcatgtacgg cagcaaaacc ttcatcaacc acacccaggg catccccgac ttctttaagc
      361 agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc
      421 tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca
      481 gaggggtgaa cttcccatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg
      541 cctccaccga gaccctgtac cccgctgacg gcggcctgga aggcagagcc gacatggccc
      601 tgaagctcgt gggcgggggc cacctgatct gcaacttgaa gaccacatac agatccaaga
      661 aacccgctaa gaacctcaag atgcccggcg tctactatgt ggacagaaga ctggaaagaa
      721 tcaaggaggc cgacaaagag acctacgtcg agcagcacga ggtggctgtg gccagatact
      781 gcgacctccc tagcaaactg gggcacagag gctccggctc cggctcccac catcaccatc
      841 accattgata agctagcacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
      901 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
      961 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     1021 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     1081 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     1141 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     1201 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     1261 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     1321 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     1381 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     1441 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     1501 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     1561 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     1621 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     1681 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     1741 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     1801 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     1861 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     1921 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     1981 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     2041 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     2101 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     2161 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     2221 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     2281 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     2341 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     2401 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2461 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2521 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2581 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2641 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2701 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2761 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2821 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     2881 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     2941 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3001 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3061 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3121 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3181 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3241 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3301 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3361 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     3421 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3481 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3541 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     3601 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     3661 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     3721 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     3781 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     3841 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     3901 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     3961 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     4021 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     4081 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     4141 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     4201 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     4261 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     4321 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     4381 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     4441 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     4501 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4561 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     4621 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     4681 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     4741 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     4801 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     4861 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     4921 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     4981 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     5041 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     5101 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     5161 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     5221 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     5281 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     5341 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     5401 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     5461 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     5521 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     5581 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     5641 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     5701 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     5761 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     5821 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     5881 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     5941 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
     6001 gaattgtgag cggataacaa ttcc
//
