LOCUS       sequence-361605-        4820 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpyTag-superfolder GFP in the bacterial cytoplasm. SpyTag 
            enables covalent reaction with SpyCatcher..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4820)
  AUTHORS   Vester SK, Rahikainen R, Khairil Anuar INA, Hills RA, Tan TK, 
            Howarth M
  TITLE     SpySwitch enables pH- or heat-responsive capture and release for 
            plug-and-display nanoassembly.
  JOURNAL   Nat Commun. 2022 Jun 28;13(1):3714. doi: 10.1038/s41467-022-31193-8.
  PUBMED    35764623
REFERENCE   2  (bases 1 to 4820)
  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 
            Commun."; date: "2022-06-28"; volume: "13(1):3714. doi"; pages: " 
            10.1038/s41467-022-31193-8"
COMMENT     Sequence Label: SpyTag-sfGFP
FEATURES             Location/Qualifiers
     source          1..4820
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      237..825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     726..745
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     979..996
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1094..1111
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1095..1111
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1567..1583
                     /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(1608..1622,1623..1628,1629..1645,1646..1652)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1628..1644
                     /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    1658..1677
                     /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             1736..1774
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     CDS             1781..1801
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1814..2524
                     /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             2543..2560
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2768..2787)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2793..2812)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2854..2945
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2946..3014,3015..3806)
                     /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(3164..3183)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             join(3889..4820,1..151)
                     /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(3908..3927)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcggtgaag ggcaatcagc tgttgccagt ctcactggtg aaaagaaaaa ccaccctggc
       61 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
      121 acaggtttcc cgactggaaa gcgggcagtg actcatgacc aaaatccctt aacgtgagtt
      181 acgcgcgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
      241 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
      301 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
      361 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttagccca ccacttcaag
      421 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
      481 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
      541 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
      601 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
      661 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
      721 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
      781 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
      841 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
      901 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
      961 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaaggcga gagtagggaa
     1021 ctgccaggca tcaaactaag cagaaggccc ctgacggatg gcctttttgc gtttctacaa
     1081 actctttctg tgttgtaaaa cgacggccag tcttaagctc gggccccctg ggcggttctg
     1141 ataacgagta atcgttaatc cgcaaataac gtaaaaaccc gcttcggcgg gtttttttat
     1201 ggggggagtt tagggaaaga gcatttgtca gaatatttaa gggcgcctgt cactttgctt
     1261 gatatatgag aattatttaa ccttataaat gagaaaaaag caacgcactt taaataagat
     1321 acgttgcttt ttcgattgat gaacacctat aattaaacta ttcatctatt atttatgatt
     1381 ttttgtatat acaatatttc tagtttgtta aagagaatta agaaaataaa tctcgaaaat
     1441 aataaaggga aaatcagttt ttgatatcaa aattatacat gtcaacgata atacaaaata
     1501 taatacaaac tataagatgt tatcagtatt tattatcatt tagaataaat tttgtgtcgc
     1561 ccttaattgt gagcggataa caattacgag cttcatgcac agtgaaatca tgaaaaattt
     1621 atttgctttg tgagcggata acaattataa tatgtggaat tgtgagcgct cacaattcca
     1681 caacggtttc cctctagaaa taattttgtt taacttttag gaggtaaaac atatggctca
     1741 tatcgtcatg gttgacgcgt ataaaccgac caaaggatca gaaaacctgt attttcaggg
     1801 aggtggttca ggccgtaaag gcgaagagct gttcactggt gtcgtcccta ttctggtgga
     1861 actggatggt gatgtcaacg gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc
     1921 aactaatggt aaactgacgc tgaagttcat ctgtactact ggtaaactgc cggtaccttg
     1981 gccgactctg gtaacgacgc tgacttatgg tgttcagtgc tttgctcgtt atccggacca
     2041 tatgaagcag catgacttct tcaagtccgc catgccggaa ggctatgtgc aggaacgcac
     2101 gatttccttt aaggatgacg gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga
     2161 taccctggta aaccgcattg agctgaaagg cattgacttt aaagaagacg gcaatatcct
     2221 gggccataag ctggaataca attttaacag ccacaatgtt tacatcaccg ccgataaaca
     2281 aaaaaatggc attaaagcga attttaaaat tcgccacaac gtggaggatg gcagcgtgca
     2341 gctggctgat cactaccagc aaaacactcc aatcggtgat ggtcctgttc tgctgccaga
     2401 caatcactat ctgagcacgc aaagcgttct gtctaaagat ccgaacgaga aacgcgatca
     2461 tatggttctg ctggagttcg taaccgcagc gggcatcacg catggtatgg atgaactgta
     2521 caaaggctcc ggctccggct cccaccatca ccatcaccat tgataactcg agccccaagg
     2581 gcgacacccc ataattagcc cgggcgaaag gcccagtctt tcgactgagc ctttcgtttt
     2641 atttgatgcc tggcagttcc ctactctcgc atggggagtc cccacactac catcggcgct
     2701 acggcgtttc acttctgagt tcggcatggg gtcaggtggg accaccgcgc tactgccgcc
     2761 aggcaaacaa ggggtgttat gagccatatt caggtataaa tgggctcgcg ataatgttca
     2821 gaattggtta attggttgta acactgaccc ctatttgttt atttttctaa atacattcaa
     2881 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2941 agaatatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     3001 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     3061 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     3121 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     3181 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     3241 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     3301 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     3361 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     3421 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     3481 cgatgcctgt agcgatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3541 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3601 tgcgctcggc ccttccggct ggctggttta ttgctgataa atccggagcc ggtgagcgtg
     3661 gttctcgcgg tatcatcgca gcgctggggc cagatggtaa gccctcccgt atcgtagtta
     3721 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     3781 gtgcctcact gattaagcat tggtaagcgg cgcgccatcg aatggcgcaa aacctttcgc
     3841 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatat gaaaccagta
     3901 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     3961 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     4021 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     4081 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     4141 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     4201 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     4261 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     4321 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgag
     4381 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     4441 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat
     4501 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc
     4561 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
     4621 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt
     4681 ggtgcggata tctcggtagt gggatacgac gataccgaag atagctcatg ttatatcccg
     4741 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
     4801 ctgcaactct ctcagggcca
//
