LOCUS       sequence-267050-        6051 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyTag003-mClover3 (a green fluorescent protein) in 
            bacterial cytoplasm.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6051)
  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 6051)
  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-mClover3
FEATURES             Location/Qualifiers
     source          1..6051
                     /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             join(117..119,120..830)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(159..180)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     767..788
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             855..872
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(942..960)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      956..1003
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1040..1495
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1127..1146)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1337..1358
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1587..2402)
                     /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(1587..2402)
                     /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     2310..2329
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2394..2413
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2524..3112
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3013..3032
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3266..3283
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3298..3437
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3363..3382)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3523..3545
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3539..3730)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4539..5621)
                     /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     5583..5602
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5622..5699)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5905..5924
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6008..6027
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6008..6026
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6027..6051
                     /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 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
      181 taaacggcca caagttcagc gtccgcggcg agggcgaggg cgatgccacc aacggcaagc
      241 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
      301 ccaccttcgg ctacggcgtg gcctgcttca gccgctaccc cgaccacatg aagcagcacg
      361 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc tctttcaagg
      421 acgacggtac ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
      481 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
      541 agtacaactt caacagccac tacgtctata tcacggccga caagcagaag aactgcatca
      601 aggctaactt caagatccgc cacaacgttg aggacggcag cgtgcagctc gccgaccact
      661 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
      721 gccatcagtc caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
      781 agttcgtgac cgccgccggg attacacatg gcatggacga gctgtacaag ggctccggct
      841 ccggctccgg ctcccaccat caccatcacc attgataagc tagcaccact gagatccggc
      901 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
      961 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     1021 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     1081 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     1141 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     1201 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     1261 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     1321 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     1381 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     1441 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     1501 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1561 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     1621 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     1681 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     1741 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     1801 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     1861 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     1921 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta
     1981 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     2041 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     2101 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     2161 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     2221 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     2281 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     2341 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     2401 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat
     2461 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2521 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2581 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2641 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     2701 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2761 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2821 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2881 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2941 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3001 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3061 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3121 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3181 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     3241 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     3301 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     3361 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     3421 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3481 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     3541 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     3601 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     3661 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     3721 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     3781 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     3841 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     3901 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     3961 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     4021 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     4081 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     4141 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     4201 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     4261 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     4321 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     4381 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     4441 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     4501 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4561 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4621 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     4681 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4741 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     4801 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4861 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     4921 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     4981 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     5041 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     5101 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     5161 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     5221 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     5281 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     5341 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     5401 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     5461 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     5521 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5581 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     5641 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     5701 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     5761 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     5821 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     5881 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     5941 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     6001 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc c
//
