LOCUS       sequence-409858-        5787 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid encoding photocaged SpyCatcher (pSC) fused to SmBiT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5787)
  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 5787)
  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: pSC-SmBiT
FEATURES             Location/Qualifiers
     source          1..5787
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     282..301
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     385..404
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        385..403
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    404..428
                     /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             443..465
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             476..814
                     /codon_start=1
                     /product="truncated version of the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyCatcher
                     /note="forms a covalent isopeptide bond with the SpyTag 
                     peptide (Zakeri et al., 2012)"
                     /translation="VDTLSGLSSEQGQSGDMTIEEDSATHIKFS*RDEDGKELAGATME
                     LRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     KATKGDAHI"
     CDS             899..916
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(969..987)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      983..1030
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1067..1522
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1154..1173)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1364..1385
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1549..1653
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1654..1722,1723..2514)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1872..1891)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2685..3273
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3174..3193
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3427..3444
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3459..3601
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3527..3546)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3687..3709
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3703..3894)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4703..5785)
                     /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     5747..5766
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(5786..5787,1..76))
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
       61 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
      121 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
      181 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
      241 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
      301 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
      361 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
      421 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac atatggttga
      481 taccttatca ggtttatcaa gtgagcaagg tcagtccggt gatatgacaa ttgaagaaga
      541 tagtgctacc catattaaat tctcatagcg tgatgaggac ggcaaagagt tagctggtgc
      601 aactatggag ttgcgtgatt catctggtaa aactattagt acatggattt cagatggaca
      661 agtgaaagat ttctacctgt atccaggaaa atatacattt gtcgaaaccg cagcaccaga
      721 cggttatgag gtagcaactg ctattacctt tacagttaat gagcaaggtc aggttactgt
      781 aaatggcaaa gcaactaaag gtgacgctca tattggtggc agtggtggtg ggagcggcgt
      841 gactggatac agactttttg aagagatcct ggaattcggg ggtggaggtt cactcgagca
      901 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
      961 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     1021 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     1081 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1141 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     1201 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1261 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     1321 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1381 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     1441 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     1501 aaaatattaa cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct
     1561 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     1621 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     1681 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     1741 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     1801 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     1861 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     1921 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     1981 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2041 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2101 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2161 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     2221 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2281 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2341 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     2401 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2461 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     2521 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     2581 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     2641 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2701 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2761 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2821 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2881 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2941 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3001 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3061 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3121 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3181 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3241 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3301 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3361 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3421 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3481 acgcatctgt gcggtatttc acaccgcata tatggtgcac tctcagtaca atctgctctg
     3541 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     3601 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3661 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3721 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     3781 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     3841 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     3901 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     3961 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     4021 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     4081 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     4141 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     4201 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     4261 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     4321 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     4381 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     4441 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     4501 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     4561 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     4621 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4681 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4741 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     4801 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     4861 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     4921 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     4981 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     5041 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     5101 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     5161 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     5221 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     5281 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     5341 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     5401 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     5461 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     5521 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     5581 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     5641 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     5701 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     5761 catcgtataa cgttactggt ttcacat
//
