LOCUS       sequence-42623-e        6484 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6484)
  AUTHORS   Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, 
            Howarth M
  TITLE     Peptide tag forming a rapid covalent bond to a protein, through 
            engineering a bacterial adhesin.
  JOURNAL   Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):E690-7. Epub 2012 Feb 
            24.
  PUBMED    22366317
REFERENCE   2  (bases 1 to 6484)
  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."; date: "2012-03-20"; volume: "109(12)"; pages: 
            "E690-7. Epub 2012 Feb 24"
COMMENT     Sequence Label: pET28a-SpyTagMBP
FEATURES             Location/Qualifiers
     source          1..6484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             8..46
                     /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             65..1162
                     /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     1136..1159
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1199..1216
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1269..1287)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1283..1330
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1367..1822
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1454..1473)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1664..1685
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1915..2730)
                     /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(1915..2730)
                     /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     2638..2657
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2722..2741
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2852..3440
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3341..3360
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3594..3611
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3626..3768
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3694..3713)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3854..3876
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3870..4061)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4870..5952)
                     /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     5914..5933
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5953..6030)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6236..6255
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6339..6358
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6339..6357
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6358..6382
                     /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             6397..6419
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6438..6455
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6465..6482
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 tatgggagcc cacatcgtga tggtggacgc ctacaagccg acgaagggta gtggtgaaag
       61 tggtaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg
      121 tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca
      181 tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat
      241 tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat
      301 caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta
      361 caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa
      421 agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact
      481 gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc
      541 gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa
      601 agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat
      661 taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa
      721 aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa
      781 agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt
      841 tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt
      901 cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct
      961 gggtgccgta gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc
     1021 cactatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt
     1081 ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga
     1141 agccctgaaa gacgcgcaga ctaattcgag ctcgtaaaag cttgcggccg cactcgagca
     1201 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     1261 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     1321 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     1381 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1441 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     1501 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1561 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     1621 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1681 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     1741 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     1801 aaaatattaa cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct
     1861 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     1921 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     1981 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     2041 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     2101 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     2161 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     2221 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     2281 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     2341 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     2401 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     2461 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     2521 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     2581 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     2641 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     2701 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     2761 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2821 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2881 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2941 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     3001 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3061 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3121 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3181 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3241 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3301 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3361 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3421 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3481 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3541 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3601 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     3661 gtatttcaca ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3721 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     3781 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3841 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3901 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     3961 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     4021 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     4081 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     4141 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     4201 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     4261 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     4321 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     4381 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     4441 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     4501 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     4561 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     4621 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     4681 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     4741 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     4801 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     4861 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4921 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     4981 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     5041 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     5101 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     5161 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     5221 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     5281 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     5341 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     5401 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     5461 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     5521 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     5581 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     5641 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     5701 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     5761 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     5821 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     5881 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     5941 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     6001 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     6061 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     6121 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     6181 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     6241 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     6301 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
     6361 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
     6421 ataccatggg cagcagccat catcatcatc atcacagcag cggcctggtg ccgcgcggca
     6481 gcca
//
