LOCUS       sequence-261758-        4281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 RNAP-driven expression of a 2-input NAND gate RNA with a GFP-ASV 
            reporter that binds to 3WJ repressor trigger indices 19 and 11.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4281)
  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 4281)
  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_NAND2_L17_S19_S11
FEATURES             Location/Qualifiers
     source          1..4281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             188..901
                     /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             188..901
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(218..246)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     841..861
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(996..1014)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1010..1057
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1287..2102)
                     /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(1287..2102)
                     /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     2010..2029
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2103..2194)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2212..2847)
                     /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(2357..2376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3045..4127)
                     /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     4089..4108
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4128..4205)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4265..4281,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4265..4281,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggactaatc agatctactt gttatagtta tgaacagagg agacataaca tgaacaagca
       61 cctaacaaga ctaatcatat acaatacctc caaatcaatc aattctactt gttatagtta
      121 tgaacagagg agacataaca tgaacaatca cataaaccta agaactaacc tggcggcagc
      181 gcaaaagatg cgtaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt
      241 agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgcaac
      301 atacggaaaa cttaccctta aatttatttg cactactgga aaactacctg ttccgtggcc
      361 aacacttgtc actactttcg gttatggtgt tcaatgcttt gcgagatacc cagatcacat
      421 gaaacagcat gactttttca agagtgccat gcccgaaggt tacgtacagg aaagaactat
      481 atttttcaaa gatgacggga actacaagac acgtgctgaa gtcaagtttg aaggtgatac
      541 ccttgttaat agaatcgagt taaaaggtat tgattttaaa gaagatggaa acattcttgg
      601 acacaaattg gaatacaact ataactcaca caatgtatac atcatggcag acaaacaaaa
      661 gaatggaatc aaagttaact tcaaaattag acacaacatt gaagatggaa gcgttcaact
      721 agcagaccat tatcaacaaa atactccgat tggcgatggc cctgtccttt taccagacaa
      781 ccattacctg tccacacaat ctgccctttc gaaagatccc aacgaaaaga gagaccacat
      841 ggtccttctt gagtttgtaa ccgctgctgg gattacacat ggcatggatg aactatacaa
      901 aaggcctgca gcaaacgacg aaaactacgc tgcatcagtt taataagata aaccagagcg
      961 gcacggcaag cagagtatac gagattcggt agccaccgct gagcaataac tagcataacc
     1021 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaacctcagg catttgagaa
     1081 gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc
     1141 ggctatttaa cgaccctgcc ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg
     1201 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     1261 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     1321 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     1381 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     1441 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     1501 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     1561 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     1621 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta
     1681 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     1741 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     1801 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     1861 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     1921 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     1981 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     2041 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     2101 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     2161 tacatatttg aatgtattta gaaaaataaa caaataggca tgctagcgca gaaacgtcct
     2221 agaagatgcc aggaggatac ttagcagaga gacaataagg ccggagcgaa gccgtttttc
     2281 cataggctcc gcccccctga cgaacatcac gaaatctgac gctcaaatca gtggtggcga
     2341 aacccgacag gactataaag ataccaggcg tttccccctg atggctccct cttgcgctct
     2401 cctgttcccg tcctgcggcg tccgtgttgt ggtggaggct ttacccaaat caccacgtcc
     2461 cgttccgtgt agacagttcg ctccaagctg ggctgtgtgc aagaaccccc cgttcagccc
     2521 gactgctgcg ccttatccgg taactatcat cttgagtcca acccggaaag acacgacaaa
     2581 acgccactgg cagcagccat tggtaactga gaattagtgg atttagatat cgagagtctt
     2641 gaagtggtgg cctaacagag gctacactga aaggacagta tttggtatct gcgctccact
     2701 aaagccagtt accaggttaa gcagttcccc aactgactta accttcgatc aaaccgcctc
     2761 cccaggcggt tttttcgttt acagagcagg agattacgac gatcgtaaaa ggatctcaag
     2821 aagatccttt acggattccc gacaccatca ctctagattt cagtgcaatt tatctcttca
     2881 aatgtagcac ctgaagtcag ccccatacga tataagttgt aattctcatg ttagtcatgc
     2941 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     3001 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     3061 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3121 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     3181 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     3241 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     3301 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
     3361 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     3421 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     3481 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     3541 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     3601 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     3661 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     3721 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     3781 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     3841 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     3901 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     3961 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     4021 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     4081 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     4141 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     4201 gtgtccggga tctcgacgct ctcccttatg gccgggttta gaaatctgaa gctctagaga
     4261 gcgctaatac gactcactat a
//
