LOCUS       sequence-269562-        4461 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 RNAP-driven expression of a 4-input NAND gate RNA with a GFP-ASV 
            reporter that binds to 3WJ repressor trigger indices 24, 11, 19, and
            13.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4461)
  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 4461)
  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_NAND4_L17_S24_S11_S19_S13
FEATURES             Location/Qualifiers
     source          1..4461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             368..1081
                     /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             368..1081
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(398..426)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1021..1041
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(1176..1194)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1190..1237
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1467..2282)
                     /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(1467..2282)
                     /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     2190..2209
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2283..2374)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2392..3027)
                     /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(2537..2556)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3225..4307)
                     /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     4269..4288
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4308..4385)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4445..4461,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4445..4461,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggctcctat cactttactt gttatagtta tgaacagagg agacataaca tgaacaagca
       61 cactaactac aaattcctaa ctttcaatcc agcatcaatc aattctactt gttatagtta
      121 tgaacagagg agacataaca tgaacaatca cataaaccta agaactaagc cgacaatact
      181 aacactaatc agatctactt gttatagtta tgaacagagg agacataaca tgaacaagca
      241 cctaacaaga ctaatcaact caatatcgca gatacaatca aatacaaatt gttatagtta
      301 tgaacagagg agacataaca tgaacaatca catacaaagc aaacgaaacc tggcggcagc
      361 gcaaaagatg cgtaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt
      421 agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgcaac
      481 atacggaaaa cttaccctta aatttatttg cactactgga aaactacctg ttccgtggcc
      541 aacacttgtc actactttcg gttatggtgt tcaatgcttt gcgagatacc cagatcacat
      601 gaaacagcat gactttttca agagtgccat gcccgaaggt tacgtacagg aaagaactat
      661 atttttcaaa gatgacggga actacaagac acgtgctgaa gtcaagtttg aaggtgatac
      721 ccttgttaat agaatcgagt taaaaggtat tgattttaaa gaagatggaa acattcttgg
      781 acacaaattg gaatacaact ataactcaca caatgtatac atcatggcag acaaacaaaa
      841 gaatggaatc aaagttaact tcaaaattag acacaacatt gaagatggaa gcgttcaact
      901 agcagaccat tatcaacaaa atactccgat tggcgatggc cctgtccttt taccagacaa
      961 ccattacctg tccacacaat ctgccctttc gaaagatccc aacgaaaaga gagaccacat
     1021 ggtccttctt gagtttgtaa ccgctgctgg gattacacat ggcatggatg aactatacaa
     1081 aaggcctgca gcaaacgacg aaaactacgc tgcatcagtt taataagata aaccagagcg
     1141 gcacggcaag cagagtatac gagattcggt agccaccgct gagcaataac tagcataacc
     1201 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaacctcagg catttgagaa
     1261 gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc
     1321 ggctatttaa cgaccctgcc ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg
     1381 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1441 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     1501 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     1561 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     1621 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     1681 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     1741 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     1801 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta
     1861 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     1921 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     1981 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     2041 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     2101 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     2161 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     2221 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     2281 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     2341 tacatatttg aatgtattta gaaaaataaa caaataggca tgctagcgca gaaacgtcct
     2401 agaagatgcc aggaggatac ttagcagaga gacaataagg ccggagcgaa gccgtttttc
     2461 cataggctcc gcccccctga cgaacatcac gaaatctgac gctcaaatca gtggtggcga
     2521 aacccgacag gactataaag ataccaggcg tttccccctg atggctccct cttgcgctct
     2581 cctgttcccg tcctgcggcg tccgtgttgt ggtggaggct ttacccaaat caccacgtcc
     2641 cgttccgtgt agacagttcg ctccaagctg ggctgtgtgc aagaaccccc cgttcagccc
     2701 gactgctgcg ccttatccgg taactatcat cttgagtcca acccggaaag acacgacaaa
     2761 acgccactgg cagcagccat tggtaactga gaattagtgg atttagatat cgagagtctt
     2821 gaagtggtgg cctaacagag gctacactga aaggacagta tttggtatct gcgctccact
     2881 aaagccagtt accaggttaa gcagttcccc aactgactta accttcgatc aaaccgcctc
     2941 cccaggcggt tttttcgttt acagagcagg agattacgac gatcgtaaaa ggatctcaag
     3001 aagatccttt acggattccc gacaccatca ctctagattt cagtgcaatt tatctcttca
     3061 aatgtagcac ctgaagtcag ccccatacga tataagttgt aattctcatg ttagtcatgc
     3121 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     3181 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     3241 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3301 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     3361 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     3421 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     3481 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
     3541 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     3601 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     3661 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     3721 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     3781 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     3841 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     3901 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     3961 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     4021 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     4081 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     4141 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     4201 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     4261 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     4321 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     4381 gtgtccggga tctcgacgct ctcccttatg gccgggttta gaaatctgaa gctctagaga
     4441 gcgctaatac gactcactat a
//
