LOCUS       sequence-141319-        5942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pGRR NOR gate promoter responsive to gRNA-r11 and gRNA-r10. 
            Integrates in the URA locus of W303 and recovers URA function..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5942)
  AUTHORS   Gander MW, Vrana JD, Voje Jr WE, Carothers JM, Klavins E
  TITLE     Robust digital logic circuits in eukaryotic cells with CRISPR/dCas9 
            NOR gates
  JOURNAL   bioRxiv 041871
REFERENCE   2  (bases 1 to 5942)
  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: "bioRxiv 
            041871"
COMMENT     Sequence Label: pMOD6-pGRR-r11-r10-yeGFP
FEATURES             Location/Qualifiers
     source          1..5942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(535..551)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(535..551)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(548..570)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    559..575
                     /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)."
     promoter        complement(join(583..589,590..607,608..613))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(766..783)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(937..1525)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(1696..2487,2488..2556))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2319..2338
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2557..2661)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2729..2747
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2785..2807)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2907..2926
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2982..3198
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             3199..4002
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLATGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     terminator      4003..4168
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4058..4082)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     4941..4957
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4941..4957
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             4964..5680
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(5682..5698)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(5712..5730)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     terminator      5742..5931
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
ORIGIN
        1 atgtcgtaat aaccccgccc cgaaaactgt attataagta aatgcatgta tactaaactc
       61 acaaattaga gcttcaattt aattatatca gttattaccc gggaatctcg gtcgtaatga
      121 tttctataat gacgaaaaaa aaaaaattgg aaagaaaaag cttcatggcc tttataaaaa
      181 ggaactatcc aatacctcgc cagaaccaag taacagtatt ttacggggca caaatcaaga
      241 acaataagac aggactgtaa agatggacgc attgaactcc aaagaacaac aagagttcca
      301 aaaagtagtg gaacaaaagc aaatgaagga tttcatgcgt ttgtactcta atctggtaga
      361 aagatgtttc acagactgtg tcaatgactt cacaacatca aagctaacca ataaggaaca
      421 aacatgcatc atgaagtgct cagaaaagtt cttgaagcat agcgaacgtg tagggcagcg
      481 tttccaagaa caaaacgctg ccttgggaca aggcttgggc cggtttaaac catggtcata
      541 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
      601 cataaagtgt aaagcctggg gtgcctaatg agtgaggtaa ctcacattaa ttgcgttgcg
      661 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
      721 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
      781 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
      841 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
      901 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
      961 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1021 ataccaggcg ttcccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1081 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     1141 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     1201 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     1261 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     1321 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     1381 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     1441 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     1501 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     1561 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     1621 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     1681 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     1741 atttcgttca tccatagttg cctgactgcc cgtcgtgtag ataactacga tacgggaggg
     1801 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     1861 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     1921 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     1981 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     2041 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     2101 gttgtgaaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     2161 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     2221 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     2281 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     2341 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     2401 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     2461 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     2521 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     2581 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     2641 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     2701 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     2761 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     2821 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     2881 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     2941 tagtttaaac tgtggctgtg gtttcagggt ccataaagct tttcaattca tctttttttt
     3001 ttttgttctt ttttttgatt ccggtttctt tgaaattttt ttgattcggt aatctccgag
     3061 cagaaggaag aacgaaggaa ggagcacaga cttagattgg tatatatacg catatgtggt
     3121 gttgaagaaa catgaaattg cccagtattc ttaacccaac tgcacagaac aaaaacctgc
     3181 aggaaacgaa gataaatcat gtcgaaagct acatataagg aacgtgctgc tactcatcct
     3241 agtcctgttg ctgccaagct atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct
     3301 tcattggatg ttcgtaccac caaggaatta ctggagttag ttgaagcatt aggtcccaaa
     3361 atttgtttac taaaaacaca tgtggatatc ttgactgatt tttccatgga gggcacagtt
     3421 aagccgctaa aggcattatc cgccaagtac aattttttac tcttcgaaga cagaaaattt
     3481 gctgacattg gtaatacagt caaattgcag tactctgcgg gtgtatacag aatagcagaa
     3541 tgggcagaca ttacgaatgc acacggtgtg gtgggcccag gtattgttag cggtttgaag
     3601 caggcggcgg aagaagtaac aaaggaacct agaggccttt tgatgttagc agaattgtca
     3661 tgcaagggct ccctagctac tggagaatat actaagggta ctgttgacat tgcgaagagc
     3721 gacaaagatt ttgttatcgg ctttattgct caaagagaca tgggtggaag agatgaaggt
     3781 tacgattggt tgattatgac acccggtgtg ggtttagatg acaagggaga cgcattgggt
     3841 caacagtata gaaccgtgga tgatgtggtc tctacaggat ctgacattat tattgttgga
     3901 agaggactat ttgcaaaggg aagggatgct aaggtagagg gtgaacgtta cagaaaagca
     3961 ggctgggaag catatttgag aagatgcggc cagcaaaact aagcgaattt cttatgattt
     4021 atgattttta ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac
     4081 tcttaggttt taaaacgaaa attcttattc ttgagtaact ctttcctgta ggtcaggttg
     4141 ctttctcagg tatagcatga ggtcgctctt attgaccaca cctcccttaa ccagattcga
     4201 aaagcggcag tttatcatta tcaatactcg ccatttcaaa gaatacgtaa ataattaata
     4261 gtagtgattt tcctaacttt atttagtcaa aaaattagcc ttttaattct gctgtaaccc
     4321 gtacatgccc aaaatagggg gcgggttaca cagaatatat aacatcgtag gtgtctgggt
     4381 gaacagttta ttcctggcat ccactaaata taatggagcc cgctttttaa gctggcatcc
     4441 agaaaaaaaa agaatcccag caccaaaata ttgttttctt caccaaccat cagttcatag
     4501 gtccattctc ttagcgcaac tacagagaac aggggcacaa acaggcaaaa aacgggcaca
     4561 acctcaatgg agtgatgcaa cctgcctgga gtaaatgatg acacaaggca attgacccac
     4621 gcatgtatct atctcatttt cttacacctt ctattacctt ctgctctctc tgatttggaa
     4681 aaagctgaaa aaaaaggttg aaaccagttc cctgaaatta ttcccctact tgactaataa
     4741 gtaaattcct gcagcccggg tactgtatgg gcaaagagac gcttgtcgtg gcatgcatgt
     4801 gctctgtatg tatataaaac tcttgttttc ttcttttctc taaatattct ttcctgagtc
     4861 gaatgtttat gtcacgcata aattactata cttctataga cacacaaaca caaatacaca
     4921 cactaatcta gatattggat tctagaacta gtggatctac aaaatgtcta aaggtgaaga
     4981 attattcact ggtgttgtcc caattttggt tgaattagat ggtgatgtta atggtcacaa
     5041 attttctgtc tccggtgaag gtgaaggtga tgctacttac ggtaaattga ccttaaaatt
     5101 tatttgtact actggtaaat tgccagttcc atggccaacc ttagtcacta ctttcggtta
     5161 tggtgttcaa tgttttgcga gatacccaga tcatatgaaa caacatgact ttttcaagtc
     5221 tgccatgcca gaaggttatg ttcaagaaag aactattttt ttcaaagatg acggtaacta
     5281 caagaccaga gctgaagtca agtttgaagg tgatacctta gttaatagaa tcgaattaaa
     5341 aggtattgat tttaaagaag atggtaacat tttaggtcac aaattggaat acaactataa
     5401 ctctcacaat gtttacatca tggctgacaa acaaaagaat ggtatcaaag ttaacttcaa
     5461 aattagacac aacattgaag atggttctgt tcaattagct gaccattatc aacaaaatac
     5521 tccaattggt gatggtccag tcttgttacc agacaaccat tacttatcca ctcaatctgc
     5581 cttatccaaa gatccaaacg aaaagagaga ccacatggtc ttgttagaat ttgttactgc
     5641 tgctggtatt acccatggta tggatgaatt gtacaaataa tgataccgtc gacctcgagt
     5701 caattagtta tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag
     5761 gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta
     5821 ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca
     5881 tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt
     5941 tg
//
