LOCUS       sequence-413107-        6671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid encoding the N-terminal UnaG fragment fused to SpyTag. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6671)
  AUTHORS   Ruskowitz ER, Munoz-Robles BG, Strange AC, Butcher CH, Kurniawan S, 
            Filteau JR, DeForest CA
  TITLE     Spatiotemporal functional assembly of split protein pairs through a 
            light-activated SpyLigation.
  JOURNAL   Nat Chem. 2023 May;15(5):694-704. doi: 10.1038/s41557-023-01152-x. 
            Epub 2023 Apr 17.
  PUBMED    37069270
REFERENCE   2  (bases 1 to 6671)
  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"; doi: 
            "10.1038/s41557-023-01152-x"; journalName: "Nat Chem"; date: 
            "2023-05"; volume: "15"; issue: "5"; pages: "694-704"
COMMENT     Sequence Label: nUnaG-ST
FEATURES             Location/Qualifiers
     source          1..6671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        106..124
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    125..149
                     /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             164..186
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             476..514
                     /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             521..1621
                     /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="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1478..1487
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1595..1618
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1637..1654
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1707..1725)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1721..1768
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1805..2260
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1892..1911)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2102..2123
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2352..3167)
                     /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(2352..3167)
                     /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     3075..3094
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3159..3178
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3289..3877
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3778..3797
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4031..4048
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4063..4202
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4128..4147)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4288..4310
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4304..4495)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5304..6386)
                     /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     6348..6367
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6387..6464)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6670..6671,1..18)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
       61 tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaattaata cgactcacta
      121 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
      181 aggagatata catatggttg aaaaatttgt gggtacgtgg aaaattgcgg attcgcataa
      241 ttttggcgaa tacctgaaag ctatcggggc acctaaagaa ctgagtgacg gaggcgatgc
      301 cacgacccca actctttata tttcccagaa ggacggtgat aaaatgaccg taaagatcga
      361 gaacggcccg cccaccttct tagatacaca agtcaaattt aaattgggtg aagaattcga
      421 tgagttcccg agcgaccgtc gcaaaggtgg ctctggaggt ggatctggcg gttctgcaca
      481 catagtaatg gtagacgcct acaagccgac gaagaagctt atgaaaatcg aagaaggtaa
      541 actggtaatc tggattaacg gcgataaagg ctataacggt ctcgctgaag tcggtaagaa
      601 attcgagaaa gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa
      661 attcccacag gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg
      721 ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca
      781 ggacaagctg tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta
      841 cccgatcgct gttgaagcgt tatcgctgat ttataacaaa gatctgctgc cgaacccgcc
      901 aaaaacctgg gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc
      961 gctgatgttc aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg
     1021 ttatgcgttc aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc
     1081 tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc
     1141 agacaccgat tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat
     1201 caacggcccg tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt
     1261 actgccgacc ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat
     1321 taacgccgcc agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac
     1381 tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc
     1441 ttacgaggaa gagttggcga aagatccacg tattgccgcc accatggaaa acgcccagaa
     1501 aggtgaaatc atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc
     1561 ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac
     1621 taattcgagc ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     1681 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1741 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     1801 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1861 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     1921 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     1981 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2041 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2101 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2161 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2221 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     2281 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2341 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     2401 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     2461 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     2521 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     2581 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     2641 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2701 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     2761 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     2821 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     2881 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     2941 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     3001 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     3061 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     3121 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     3181 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     3241 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3301 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3361 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3421 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3481 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3541 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3601 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3661 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3721 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3781 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3841 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3901 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3961 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4021 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     4081 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     4141 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     4201 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4261 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4321 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     4381 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     4441 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     4501 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     4561 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     4621 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     4681 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     4741 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     4801 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     4861 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     4921 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     4981 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     5041 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     5101 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     5161 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     5221 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     5281 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5341 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     5401 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     5461 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     5521 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     5581 gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     5641 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     5701 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     5761 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     5821 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5881 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5941 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     6001 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     6061 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     6121 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     6181 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     6241 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     6301 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     6361 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     6421 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     6481 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     6541 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     6601 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     6661 ttccccatcg g
//
