LOCUS       sequence-267047-        6042 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyTag003-superfolder GFP in bacterial cytoplasm.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6042)
  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 6042)
  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-sfGFP
FEATURES             Location/Qualifiers
     source          1..6042
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             14..61
                     /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             80..790
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="RKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             809..826
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(896..914)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      910..957
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      994..1449
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1081..1100)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1291..1312
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1541..2356)
                     /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(1541..2356)
                     /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     2264..2283
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2348..2367
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2478..3066
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2967..2986
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3220..3237
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3252..3391
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3317..3336)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3477..3499
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3493..3684)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4493..5575)
                     /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     5537..5556
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5576..5653)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5859..5878
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5962..5981
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5962..5980
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5981..6005
                     /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             6020..6042
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 tatacatatg ggacgtggcg ttcctcatat tgttatggtg gacgcctaca aacgctataa
       61 atcgggaggt ggttcaggcc gtaaaggcga agagctgttc actggtgtcg tccctattct
      121 ggtggaactg gatggtgatg tcaacggtca taagttttcc gtgcgtggcg agggtgaagg
      181 tgacgcaact aatggtaaac tgacgctgaa gttcatctgt actactggta aactgccggt
      241 accttggccg actctggtaa cgacgctgac ttatggtgtt cagtgctttg ctcgttatcc
      301 ggaccatatg aagcagcatg acttcttcaa gtccgccatg ccggaaggct atgtgcagga
      361 acgcacgatt tcctttaagg atgacggcac gtacaaaacg cgtgcggaag tgaaatttga
      421 aggcgatacc ctggtaaacc gcattgagct gaaaggcatt gactttaaag aagacggcaa
      481 tatcctgggc cataagctgg aatacaattt taacagccac aatgtttaca tcaccgccga
      541 taaacaaaaa aatggcatta aagcgaattt taaaattcgc cacaacgtgg aggatggcag
      601 cgtgcagctg gctgatcact accagcaaaa cactccaatc ggtgatggtc ctgttctgct
      661 gccagacaat cactatctga gcacgcaaag cgttctgtct aaagatccga acgagaaacg
      721 cgatcatatg gttctgctgg agttcgtaac cgcagcgggc atcacgcatg gtatggatga
      781 actgtacaaa ggctccggct ccggctccca ccatcaccat caccattgat aagctagcac
      841 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
      901 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
      961 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     1021 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1081 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1141 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1201 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1261 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1321 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1381 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1441 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1501 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     1561 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1621 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     1681 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     1741 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     1801 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     1861 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     1921 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     1981 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     2041 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     2101 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     2161 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     2221 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     2281 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     2341 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     2401 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2461 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2521 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2581 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     2641 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2701 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2761 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2821 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2881 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2941 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     3001 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3061 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3121 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3181 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3241 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3301 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     3361 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     3421 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3481 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     3541 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     3601 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     3661 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     3721 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     3781 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     3841 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     3901 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     3961 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     4021 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     4081 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     4141 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     4201 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     4261 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4321 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4381 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     4441 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     4501 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4561 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     4621 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     4681 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     4741 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     4801 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     4861 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     4921 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     4981 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     5041 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     5101 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     5161 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     5221 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     5281 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     5341 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     5401 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     5461 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     5521 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     5581 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     5641 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     5701 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     5761 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     5821 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     5881 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     5941 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     6001 attcccctct agaaataatt ttgtttaact ttaagaagga ga
//
