LOCUS       sequence-413108-        5780 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid encoding LgBiT fused to SpyTag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5780)
  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 5780)
  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: LgBiT-ST
FEATURES             Location/Qualifiers
     source          1..5780
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             519..557
                     /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             564..581
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(634..652)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      648..695
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      732..1187
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(819..838)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1029..1050
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1279..2094)
                     /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(1279..2094)
                     /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     2002..2021
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2086..2105
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2216..2804
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2705..2724
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2958..2975
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2990..3129
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3055..3074)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3215..3237
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3231..3422)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4231..5313)
                     /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     5275..5294
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5314..5391)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5597..5616
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5704..5723
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5704..5722
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5723..5747
                     /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             join(5762..5780,1..4)
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 gagatataca tatggtcttc acactcgaag atttcgttgg ggactgggaa cagacagccg
       61 cgtacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgctg cagaatctcg
      121 cggtgtccgt aactccgatc caaaggattg tccgcagcgg tgaaaatgcg ctgaagatcg
      181 acatccatgt catcatcccg tatgaaggtc tgagcgcgga ccaaatggca cagatcgaag
      241 aagtgtttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc ctgccgtatg
      301 gcacactggt aatcgacggg gttacgccga acatgctgaa ctatttcgga cggccgtatg
      361 aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg tggaacggca
      421 acaaaattat cgacgagcgc ctgatcaccc ccgacggctc catgctgttc cgagtaacca
      481 tcaacagcgg tggctctgga ggtggatctg gcggttctgc acacatagta atggtagacg
      541 cctacaagcc gacgaagctc gagcaccacc accaccacca ctgagatccg gctgctaaca
      601 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
      661 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
      721 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
      781 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      841 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      901 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      961 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1021 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     1081 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     1141 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     1201 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1261 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     1321 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     1381 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     1441 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     1501 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     1561 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     1621 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     1681 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1741 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1801 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1861 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1921 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1981 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     2041 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     2101 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     2161 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2221 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2281 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2341 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     2401 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2461 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2521 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2581 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2641 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2701 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2761 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2821 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2881 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2941 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     3001 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     3061 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     3121 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3181 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3241 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     3301 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     3361 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     3421 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     3481 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     3541 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     3601 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     3661 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3721 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3781 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     3841 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     3901 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     3961 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     4021 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     4081 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     4141 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     4201 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4261 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4321 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4381 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4441 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4501 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4561 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     4621 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     4681 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4741 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4801 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4861 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4921 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4981 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5041 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     5101 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     5161 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     5221 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5281 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5341 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5401 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     5461 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     5521 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     5581 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     5641 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatcgatctc gatcccgcga
     5701 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     5761 ttttgtttaa ctttaagaag
//
