LOCUS       sequence-486522-        6123 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses multifunctional construct enabling the YES-gated release 
            of mCherry from materials in response to sortase eSrtA(4S9) 
            treatment. Construct contains a SpyTag for material tethering..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6123)
  AUTHORS   Gharios R, Ross ML, Li A, Kottantharayil SP, Hoye J, DeForest CA
  TITLE     Boolean logic-gated protein presentation through autonomously 
            compiled molecular topology.
  JOURNAL   Nat Chem Biol. 2025 Oct 9. doi: 10.1038/s41589-025-02037-5.
  PUBMED    41068299
REFERENCE   2  (bases 1 to 6123)
  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: "Nat Chem 
            Biol. 2025 Oct 9. doi: 10.1038/s41589-025-02037-5."
COMMENT     Sequence Label: mCherry-S-SpyTag
FEATURES             Location/Qualifiers
     source          1..6123
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(167..184)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(212..259)
                     /codon_start=1
                     /product="improved SpyTag variant that reacts with 
                     SpyCatcher003 at a rate approaching the diffusion limit 
                     (Keeble et al., 2019)"
                     /label=SpyTag003
                     /translation="RGVPHIVMVDAYKRYK"
     RBS             1056..1078
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1093..1117
                     /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)."
     primer_bind     complement(1117..1136)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1118..1136)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1224..1243)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1449..1526
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1527..2609
                     /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     complement(1546..1565)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3418..3609
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3603..3625)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3711..3853
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3766..3785
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3868..3885)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4039..4627)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4119..4138)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4738..4757)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4749..5564
                     /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             4749..5564
                     /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     complement(4822..4841)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5657..6112)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5794..5815)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6006..6025
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagttaatga tggtgatgat
      181 ggtgtgagcc gccgctgccg ccggaaccac ccttgtagcg cttataggca tcaaccataa
      241 caatatgcgg tacgccccga ctgccgccgg aacctcctga acctccgccg ctttccggca
      301 ggcttccacc actaccgccg gaaccgccct tgtacagctc gtccatgccg cccgtagagt
      361 ggcgaccttc ggcacgttcg tactgttcca ctatggtata atcttcatta tgagatgtga
      421 tgtcaagttt aatattaacg ttatatgcac ccggcagctg cactggtttt ttcgccttat
      481 aagtggtttt cacctccgca tcataatgtc cgccatcttt gagtttcaga cgttgtttaa
      541 tttccccttt caatgcccca tcttcggggt acatacgttc gctagacgct tcccaaccca
      601 tggttttttt ttgcatgacc gggccgtccg acggaaagtt agtcccgcgc agtttgactt
      661 tataaataaa ttccccatcc tgcagcgacg aatcctgcgt gactgtgacc acgcccccat
      721 cctcgaagtt catcactcgc tcccatttaa aaccctcagg aaacgacaac tttaaatagt
      781 ctgggatatc tgccgggtgt ttaacgtacg ccttgctacc atacataaat tgcggactta
      841 gaatgtccca ggcgaagggt aaaggtccgc ctttggttac tttcagttta gcggtctgcg
      901 taccttcgta tgggcgaccc tcgccttcgc cttcgatctc gaattcgtga ccgttaacgc
      961 ttccttccat atgtactttg aaacgcataa attctttaat gatagccata ttgtcctctt
     1021 cccctttgga caccattgaa ctccccatat gtatatctcc ttcttaaagt taaacaaaat
     1081 tatttctaga ggggaattgt tatccgctca caattcccct atagtgagtc gtattaattt
     1141 cgcgggatcg agatcgatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg
     1201 cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc
     1261 tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc
     1321 cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa
     1381 cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg
     1441 agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
     1501 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
     1561 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
     1621 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
     1681 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
     1741 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
     1801 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
     1861 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
     1921 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
     1981 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
     2041 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
     2101 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
     2161 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
     2221 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     2281 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggacatc tcggtagtgg
     2341 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     2401 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     2461 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     2521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact
     2641 cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc
     2701 cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa
     2761 ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg
     2821 agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa
     2881 gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc
     2941 atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg
     3001 cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac
     3061 tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt
     3121 tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc
     3181 gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat
     3241 tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca
     3301 caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct
     3361 cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg
     3421 accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg
     3481 cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt
     3541 cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa
     3601 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     3661 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     3721 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     3781 ttgtactgag agtgcaccat atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     3841 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3901 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3961 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4021 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4081 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4141 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4201 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4261 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4321 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4381 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4441 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     4501 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4561 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4621 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4681 acgttaaggg attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     4741 ggggtgttat gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca
     4801 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     4861 cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     4921 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     4981 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     5041 ccactgcgat ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     5101 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     5161 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     5221 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     5281 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     5341 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     5401 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     5461 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     5521 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc
     5581 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5641 cgaaaagtgc cacctgaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt
     5701 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     5761 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     5821 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     5881 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     5941 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     6001 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     6061 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
     6121 cca
//
