LOCUS       sequence-201087-        6474 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of unreactive version of SpyTag002 fused to 
            maltose binding protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6474)
  AUTHORS   Keeble AH, Banerjee A, Reddington SC, Ferla MP, Howarth M, Khairil 
            Anuar INA
  TITLE     Evolving accelerated amidation by SpyTag/SpyCatcher to analyze 
            membrane dynamics.
  JOURNAL   Angew Chem Int Ed Engl. 2017 Oct 10. doi: 10.1002/anie.201707623.
  PUBMED    29024296
REFERENCE   2  (bases 1 to 6474)
  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: "Angew Chem 
            Int Ed Engl. 2017 Oct 10. doi: 10.1002/anie.201707623."
COMMENT     Sequence Label: pET28a-SpyTag002 DA-MBP
FEATURES             Location/Qualifiers
     source          1..6474
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             145..186
                     /codon_start=1
                     /product="improved SpyTag variant that shows rapid reaction
                     with SpyCatcher002 (Keeble et al., 2017)"
                     /label=SpyTag002
                     /translation="VPTIVMVAAYKRYK"
     CDS             205..1302
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1276..1299
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1339..1356
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1409..1427)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1423..1470
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1507..1962
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1594..1613)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1804..1825
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2054..2869)
                     /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(2054..2869)
                     /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     2777..2796
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2861..2880
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2991..3579
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3480..3499
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3733..3750
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3765..3904
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3830..3849)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3990..4012
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4006..4197)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5006..6088)
                     /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     6050..6069
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6089..6166)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6372..6391
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagcgtgcct actatcgtga tggtggccgc ctacaagcgt
      181 tacaagggta gtggtgaaag tggtaaaatc gaagaaggta aactggtaat ctggattaac
      241 ggcgataaag gctataacgg tctcgctgaa gtcggtaaga aattcgagaa agataccgga
      301 attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca ggttgcggca
      361 actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg ctacgctcaa
      421 tctggcctgt tggctgaaat caccccggac aaagcgttcc aggacaagct gtatccgttt
      481 acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc tgttgaagcg
      541 ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctg ggaagagatc
      601 ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa
      661 gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt caagtatgaa
      721 aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa agcgggtctg
      781 accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga ttactccatc
      841 gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg
      901 tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgac cttcaagggt
      961 caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac
     1021 aaagagctgg caaaagagtt cctcgaaaac tatctgctga ctgatgaagg tctggaagcg
     1081 gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga agagttggcg
     1141 aaagatccac gtattgccgc cactatggaa aacgcccaga aaggtgaaat catgccgaac
     1201 atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc
     1261 ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag ctcgtaaaag
     1321 cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     1381 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     1441 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     1501 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1561 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1621 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     1681 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     1741 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     1801 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     1861 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     1921 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     1981 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     2041 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     2101 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     2161 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     2221 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     2281 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
     2341 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     2401 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
     2461 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     2521 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     2581 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     2641 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     2701 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     2761 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     2821 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
     2881 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
     2941 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3001 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3061 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3121 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     3181 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3241 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3301 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3361 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3421 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3481 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3541 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3601 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3661 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3721 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     3781 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     3841 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     3901 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3961 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4021 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     4081 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     4141 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     4201 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     4261 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     4321 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     4381 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     4441 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     4501 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     4561 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     4621 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     4681 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     4741 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     4801 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     4861 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     4921 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     4981 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5041 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     5101 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     5161 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     5221 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     5281 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     5341 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     5401 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     5461 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     5521 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     5581 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     5641 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     5701 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     5761 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     5821 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     5881 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     5941 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     6001 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     6061 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     6121 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     6181 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     6241 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     6301 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     6361 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     6421 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaat
//
