LOCUS       sequence-261760-        4359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 RNAP-driven expression of a 3-input NAND gate RNA with a GFP-ASV 
            reporter that binds to 3WJ repressor trigger indices 11, 13, and 19.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4359)
  AUTHORS   Kim J, Zhou Y, Carlson PD, Teichmann M, Chaudhary S, Simmel FC, 
            Silver PA, Collins JJ, Lucks JB, Yin P, Green AA
  TITLE     De novo-designed translation-repressing riboregulators for 
            multi-input cellular logic.
  JOURNAL   Nat Chem Biol. 2019 Dec;15(12):1173-1182. doi: 
            10.1038/s41589-019-0388-1. Epub 2019 Nov 4.
  PUBMED    31686032
REFERENCE   2  (bases 1 to 4359)
  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/s41589-019-0388-1"; journalName: "Nat Chem Biol"; date: 
            "2019-12"; volume: "15"; issue: "12"; pages: "1173-1182"
COMMENT     Sequence Label: pYZ_NAND3_L11_S11_S13_S19
FEATURES             Location/Qualifiers
     source          1..4359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             266..979
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             266..979
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(296..324)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     919..939
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(1074..1092)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1088..1135
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1365..2180)
                     /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(1365..2180)
                     /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     2088..2107
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2181..2272)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2290..2925)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     complement(2435..2454)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3123..4205)
                     /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     4167..4186
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4206..4283)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4343..4359,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4343..4359,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggatcaatc aattctactt gttatagtta tgaacagagg agacataaca tgaacaatca
       61 cataaaccta agaactcaat atcgcataca atcaaataca aattgttata gttatgaaca
      121 gaggagacat aacatgaaca atcacataca aagcaaacga caaatacaaa tactaatcag
      181 atctacttgt tatagttatg aacagaggag acataacatg aacaagcacc taacaagact
      241 aatcaacctg gcggcagcgc aaaagatgcg taaaggagaa gaacttttca ctggagttgt
      301 cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga
      361 gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca ctactggaaa
      421 actacctgtt ccgtggccaa cacttgtcac tactttcggt tatggtgttc aatgctttgc
      481 gagataccca gatcacatga aacagcatga ctttttcaag agtgccatgc ccgaaggtta
      541 cgtacaggaa agaactatat ttttcaaaga tgacgggaac tacaagacac gtgctgaagt
      601 caagtttgaa ggtgataccc ttgttaatag aatcgagtta aaaggtattg attttaaaga
      661 agatggaaac attcttggac acaaattgga atacaactat aactcacaca atgtatacat
      721 catggcagac aaacaaaaga atggaatcaa agttaacttc aaaattagac acaacattga
      781 agatggaagc gttcaactag cagaccatta tcaacaaaat actccgattg gcgatggccc
      841 tgtcctttta ccagacaacc attacctgtc cacacaatct gccctttcga aagatcccaa
      901 cgaaaagaga gaccacatgg tccttcttga gtttgtaacc gctgctggga ttacacatgg
      961 catggatgaa ctatacaaaa ggcctgcagc aaacgacgaa aactacgctg catcagttta
     1021 ataagataaa ccagagcggc acggcaagca gagtatacga gattcggtag ccaccgctga
     1081 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     1141 acctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa
     1201 ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga caagctgacg
     1261 accgggtctc cgcaagtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1321 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     1381 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     1441 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     1501 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     1561 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     1621 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     1681 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     1741 aatacgcggt cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     1801 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     1861 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     1921 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     1981 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     2041 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     2101 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     2161 tcccgttgaa tatggctcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     2221 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggcatg
     2281 ctagcgcaga aacgtcctag aagatgccag gaggatactt agcagagaga caataaggcc
     2341 ggagcgaagc cgtttttcca taggctccgc ccccctgacg aacatcacga aatctgacgc
     2401 tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgat
     2461 ggctccctct tgcgctctcc tgttcccgtc ctgcggcgtc cgtgttgtgg tggaggcttt
     2521 acccaaatca ccacgtcccg ttccgtgtag acagttcgct ccaagctggg ctgtgtgcaa
     2581 gaaccccccg ttcagcccga ctgctgcgcc ttatccggta actatcatct tgagtccaac
     2641 ccggaaagac acgacaaaac gccactggca gcagccattg gtaactgaga attagtggat
     2701 ttagatatcg agagtcttga agtggtggcc taacagaggc tacactgaaa ggacagtatt
     2761 tggtatctgc gctccactaa agccagttac caggttaagc agttccccaa ctgacttaac
     2821 cttcgatcaa accgcctccc caggcggttt tttcgtttac agagcaggag attacgacga
     2881 tcgtaaaagg atctcaagaa gatcctttac ggattcccga caccatcact ctagatttca
     2941 gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata taagttgtaa
     3001 ttctcatgtt agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     3061 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     3121 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3181 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     3241 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     3301 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     3361 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc
     3421 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     3481 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     3541 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     3601 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     3661 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     3721 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     3781 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     3841 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     3901 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     3961 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     4021 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     4081 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     4141 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     4201 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     4261 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatggc cgggtttaga
     4321 aatctgaagc tctagagagc gctaatacga ctcactata
//
