LOCUS       sequence-494257-        6138 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses multifunctional construct enabling the YES-gated release 
            of mCerulean from materials in response to TEV treatment. Construct 
            contains a SpyTag for material tethering..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6138)
  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 6138)
  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: mCerulean-T-SpyTag
FEATURES             Location/Qualifiers
     source          1..6138
                     /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"
     CDS             complement(287..307)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     RBS             1071..1093
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1108..1132
                     /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(1132..1151)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1133..1151)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1239..1258)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1464..1541
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1542..2624
                     /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(1561..1580)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3433..3624
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3618..3640)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3726..3868
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3781..3800
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3883..3900)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4054..4642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4134..4153)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4753..4772)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4764..5579
                     /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             4764..5579
                     /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(4837..4856)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5672..6127)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5809..5830)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6021..6040
                     /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 gatggctacc accagaccca ccactgccac ctttatatcg cttgtatgca tcaaccataa
      241 caatatgcgg tacaccgcgg ctgcctccag accccccgga gccgcctgac tgaaagtaca
      301 gattttcgct gcccccggaa ccacctgatc cacctttgta cagttcatcc atacctaaag
      361 taatgcccgc ggcggtgacg aactccagca acaccatgtg gtcgcgtttt tcgtttgggt
      421 cctttgaaag tttactttga gtacttaaat agtgattatc aggaagcagg acaggaccgt
      481 cgccgatggg ggtattttgt tggtagtgat ccgcgagctg cacgcttcca tcttcaatgt
      541 tatggcggat tttaaagttg gccttaatgc cgttcttctg tttgtctgct gtaatataaa
      601 cgttatccga aatcgcgtta tactccaatt tatgaccgag tatgttccca tcctctttga
      661 aatcgatccc tttcagttcg atacgattaa ccagagtatc accctcaaat ttaacttcgg
      721 cacgcgtttt ataattccca tcgtctttaa aaaagattgt ccgttcctgc acgtagccct
      781 ccggcatagc gcttttaaag aagtcgtgct gtttcatgtg atccggataa cgagcaaagc
      841 actgtacgcc ccacgtcagg gtggtcacca gcgttggcca cggcaccggt agttttcccg
      901 tggtacagat gaatttcagg gttaacttgc cataggtcgc atcgccttcg ccttcgccgc
      961 tcaccgaaaa cttatggcca tttacgtcac catccaattc caccagaatc gggaccacac
     1021 ccgtgaacag ttcttcgcct ttggagacca tcgaagatcc catatgtata tctccttctt
     1081 aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt
     1141 gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc ctctacgccg gacgcatcgt
     1201 ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga
     1261 tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt
     1321 ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc
     1381 ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca
     1441 taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg
     1501 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     1561 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     1621 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     1681 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     1741 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     1801 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     1861 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     1921 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     1981 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     2041 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     2101 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     2161 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     2221 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     2281 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     2341 acatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     2401 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     2461 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     2521 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     2581 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     2641 gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac
     2701 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc
     2761 ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag
     2821 gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg
     2881 cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag
     2941 gccattatcg ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg
     3001 acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag
     3061 cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc
     3121 ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg
     3181 ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta
     3241 acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc
     3301 agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc
     3361 gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac
     3421 acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga
     3481 agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac
     3541 atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc
     3601 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     3661 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     3721 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg
     3781 cggcatcaga gcagattgta ctgagagtgc accatatatg cggtgtgaaa taccgcacag
     3841 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     3901 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3961 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4021 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4081 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4141 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4201 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4261 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4321 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4381 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4441 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     4501 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4561 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4621 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4681 gtggaacgaa aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca
     4741 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc
     4801 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg
     4861 tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt
     4921 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa
     4981 actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg
     5041 atgcatggtt actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat
     5101 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt
     5161 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc
     5221 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     5281 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag
     5341 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     5401 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat
     5461 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     5521 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag
     5581 aattaattca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5641 ccgcgcacat ttccccgaaa agtgccacct gaaattgtaa acgttaatat tttgttaaaa
     5701 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     5761 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     5821 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     5881 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     5941 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     6001 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     6061 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     6121 ggcgcgtccc attcgcca
//
