LOCUS       sequence-266482-        5136 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyCatcher003-superfolder GFP in bacterial cytoplasm.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5136)
  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 5136)
  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: SpyCatcher003-sfGFP
FEATURES             Location/Qualifiers
     source          1..5136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     127..144
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     242..259
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     243..259
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    715..731
                     /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)."
     promoter        join(756..770,771..776,777..793,794..800)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    776..792
                     /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)."
     protein_bind    806..825
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     CDS             894..911
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             933..953
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             960..1298
                     /codon_start=1
                     /product="improved SpyCatcher variant that reacts with 
                     SpyTag003 at a rate approaching the diffusion limit (Keeble
                     et al., 2019)"
                     /label=SpyCatcher003
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDG
                     EATEGDAHT"
     CDS             1314..2024
                     /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"
     primer_bind     complement(2232..2251)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2257..2276)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2318..2409
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2410..2478,2479..3270)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2628..2647)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             3353..4435
                     /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     complement(3372..3391)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      4521..5109
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5010..5029
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
       61 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
      121 gagcgcagcg agtcagtgag cgaggaagcg gaaggcgaga gtagggaact gccaggcatc
      181 aaactaagca gaaggcccct gacggatggc ctttttgcgt ttctacaaac tctttctgtg
      241 ttgtaaaacg acggccagtc ttaagctcgg gccccctggg cggttctgat aacgagtaat
      301 cgttaatccg caaataacgt aaaaacccgc ttcggcgggt ttttttatgg ggggagttta
      361 gggaaagagc atttgtcaga atatttaagg gcgcctgtca ctttgcttga tatatgagaa
      421 ttatttaacc ttataaatga gaaaaaagca acgcacttta aataagatac gttgcttttt
      481 cgattgatga acacctataa ttaaactatt catctattat ttatgatttt ttgtatatac
      541 aatatttcta gtttgttaaa gagaattaag aaaataaatc tcgaaaataa taaagggaaa
      601 atcagttttt gatatcaaaa ttatacatgt caacgataat acaaaatata atacaaacta
      661 taagatgtta tcagtattta ttatcattta gaataaattt tgtgtcgccc ttaattgtga
      721 gcggataaca attacgagct tcatgcacag tgaaatcatg aaaaatttat ttgctttgtg
      781 agcggataac aattataata tgtggaattg tgagcgctca caattccaca acggtttccc
      841 tgtagaaata attttgttta actttaataa ggagatatac catgtcgtac taccatcacc
      901 atcaccatca cgattacgac atcccaacga ccgaaaacct gtattttcag ggcgccatgg
      961 taaccacctt atcaggttta tcaggtgagc aaggtccgtc cggtgatatg acaactgaag
     1021 aagatagtgc tacccatatt aaattctcaa aacgtgatga ggacggccgt gagttagctg
     1081 gtgcaactat ggagttgcgt gattcatctg gtaaaactat tagtacatgg atttcagatg
     1141 gacatgtgaa ggatttctac ctgtatccag gaaaatatac atttgtcgaa accgcagcac
     1201 cagacggtta tgaggtagca actccaattg aatttacagt taatgaggac ggtcaggtta
     1261 ctgtagatgg tgaagcaact gaaggtgacg ctcatactgg atccagtggt agccgtaaag
     1321 gcgaagagct gttcactggt gtcgtcccta ttctggtgga actggatggt gatgtcaacg
     1381 gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc aactaatggt aaactgacgc
     1441 tgaagttcat ctgtactact ggtaaactgc cggtaccttg gccgactctg gtaacgacgc
     1501 tgacttatgg tgttcagtgc tttgctcgtt atccggacca tatgaagcag catgacttct
     1561 tcaagtccgc catgccggaa ggctatgtgc aggaacgcac gatttccttt aaggatgacg
     1621 gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga taccctggta aaccgcattg
     1681 agctgaaagg cattgacttt aaagaagacg gcaatatcct gggccataag ctggaataca
     1741 attttaacag ccacaatgtt tacatcaccg ccgataaaca aaaaaatggc attaaagcga
     1801 attttaaaat tcgccacaac gtggaggatg gcagcgtgca gctggctgat cactaccagc
     1861 aaaacactcc aatcggtgat ggtcctgttc tgctgccaga caatcactat ctgagcacgc
     1921 aaagcgttct gtctaaagat ccgaacgaga aacgcgatca tatggttctg ctggagttcg
     1981 taaccgcagc gggcatcacg catggtatgg atgaactgta caaatgataa ctcgagcccc
     2041 aagggcgaca ccccataatt agcccgggcg aaaggcccag tctttcgact gagcctttcg
     2101 ttttatttga tgcctggcag ttccctactc tcgcatgggg agtccccaca ctaccatcgg
     2161 cgctacggcg tttcacttct gagttcggca tggggtcagg tgggaccacc gcgctactgc
     2221 cgccaggcaa acaaggggtg ttatgagcca tattcaggta taaatgggct cgcgataatg
     2281 ttcagaattg gttaattggt tgtaacactg acccctattt gtttattttt ctaaatacat
     2341 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2401 aggaagaata tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2461 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2521 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2581 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2641 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2701 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2761 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2821 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2881 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2941 accacgatgc ctgtagcgat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3001 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3061 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatccgg agccggtgag
     3121 cgtggttctc gcggtatcat cgcagcgctg gggccagatg gtaagccctc ccgtatcgta
     3181 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3241 ataggtgcct cactgattaa gcattggtaa gcggcgcgcc atcgaatggc gcaaaacctt
     3301 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atatgaaacc
     3361 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     3421 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     3481 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     3541 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     3601 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     3661 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     3721 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     3781 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     3841 tgaggacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     3901 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     3961 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     4021 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     4081 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     4141 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagatagct catgttatat
     4201 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     4261 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc cagtctcact
     4321 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     4381 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgactcat
     4441 gaccaaaatc ccttaacgtg agttacgcgc gcgtcgttcc actgagcgtc agaccccgta
     4501 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4561 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4621 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     4681 ccgtagttag cccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4741 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4801 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4861 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4921 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4981 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     5041 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     5101 ctatggaaaa acgccagcaa cgcggccttt ttacgg
//
