LOCUS       sequence-141329-        6590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid constitutively expresses gRNA-r10 from an AHD1 promoter
            using the RGR design. Integrates in the W303 HIS locus and restores 
            HIS function..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6590)
  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 6590)
  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: pMOD8A-RGR-r10
FEATURES             Location/Qualifiers
     source          1..6590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1036..1052)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1036..1052)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1049..1071)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1060..1076
                     /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(1084..1090,1091..1108,1109..1114))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1129..1150
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1267..1284)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1438..2026)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1518..1537)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2197..2988,2989..3057))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2820..2839
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3058..3162)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3230..3248
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3286..3308)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3408..3427
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3580..3767
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             3768..4427
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
                     ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     terminator      4428..4593
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4483..4507)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        5374..6078
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     primer_bind     6095..6111
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6095..6111
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     ncRNA           6124..6160
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        6181..6256
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           6261..6328
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(6330..6346)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(6360..6378)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     terminator      6390..6579
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
ORIGIN
        1 atgtcgtaat aaccccgccc cgtgacaccg attatttaaa gctgcagcat acgatatata
       61 tacatgtgta tatatgtata cctatgaatg tcagtaagta tgtatacgaa cagtatgata
      121 ctgaagatga caaggtaatg catcattcta tacgtgtcat tctgaacgag gcgcgctttc
      181 cttttttctt tttgcttttt cttttttttt ctcttgaact cgagaaaaaa aatataaaag
      241 agatggagga acgggaaaaa gttagttgtg gtgataggtg gcaagtggta ttccgtaaga
      301 acaacaagaa aagcatttca tattatggct gaactgagcg aacaagtgca aaatttaagc
      361 atcaacgaca acaacgagaa tggttatgtt cctcctcact taagaggaaa accaagaagt
      421 gccagaaata acagtagcaa ctacaataac aacaacggcg gctacaacgg tggccgtggc
      481 ggtggcagct tctttagcaa caaccgtcgt ggtggttacg gcaacggtgg tttcttcggt
      541 ggaaacaacg gtggcagcag atctaacggc cgttctggtg gtagatggat cgatggcaaa
      601 catgtcccag ctccaagaaa cgaaaaggcc gagatcgcca tatttggtgt ccccgaggat
      661 ccaaatttcc aatcttctgg tattaacttc gataactacg atgatattcc agtggacgcc
      721 tctggtaagg atgttcctga accaatcaca gaatttacct cacctccatt ggacggattg
      781 ttattggaaa acatcaaatt ggcccgtttc accaagccaa cacctgtgca aaaatactcc
      841 gtccctatcg ttgccaacgg cagagatttg atggcctgtg cgcagaccgg ttctggtaag
      901 actggtgggt ttttattccc agtgttgtcc gaatcattta agactggacc atctcctcaa
      961 ccagagtctc aaggctcctt ttaccaaaga aaggcctacc caactgctgt cattatggct
     1021 ccagtttaaa ccatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     1081 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     1141 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     1201 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     1261 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     1321 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     1381 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     1441 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     1501 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     1561 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     1621 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     1681 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     1741 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     1801 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     1861 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     1921 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     1981 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2041 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     2101 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     2161 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     2221 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     2281 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     2341 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     2401 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     2461 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     2521 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     2581 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     2641 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     2701 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     2761 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     2821 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     2881 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     2941 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     3001 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     3061 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     3121 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     3181 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     3241 cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     3301 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     3361 gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca
     3421 gattgtactg agagtgcacc ataattcgtt taaaccgttt taagagcttg gtgagcgcta
     3481 ggagtcactg ccaggtatcg tttgaacacg gcattagtca gggaagtcat aacacagtcc
     3541 tttcccgcaa ttttcttttt ctattactct tggcctcctc tagtacactc tatatttttt
     3601 tatgcctcgg taatgatttt catttttttt tttccaccta gcggatgact cttttttttt
     3661 cttagcgatt ggcattatca cataatgaat tatacattat ataaagtaat gtgatttctt
     3721 cgaagaatat actaaaaaat gagcaggcaa gataaacgaa ggcaaagatg acagagcaga
     3781 aagccctagt aaagcgtatt acaaatgaaa ccaagattca gattgcgatc tctttaaagg
     3841 gtggtcccct agcgatagag cactcgatct tcccagaaaa agaggcagaa gcagtagcag
     3901 aacaggccac acaatcgcaa gtgattaacg tccacacagg tatagggttt ctggaccata
     3961 tgatacatgc tctggccaag cattccggct ggtcgctaat cgttgagtgc attggtgact
     4021 tacacataga cgaccatcac accactgaag actgcgggat tgctctcggt caagctttta
     4081 aagaggccct actggcgcgt ggagtaaaaa ggtttggatc aggatttgcg cctttggatg
     4141 aggcactttc cagagcggtg gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg
     4201 gtttgcaaag ggagaaagta ggagatctct cttgcgagat gatcccgcat tttcttgaaa
     4261 gctttgcaga ggctagcaga attaccctcc acgttgattg tctgcgaggc aagaatgatc
     4321 atcaccgtag tgagagtgcg ttcaaggctc ttgcggttgc cataagagaa gccacctcgc
     4381 ccaatggtac caacgatgtt ccctccacca aaggtgttct tatgtaggcg aatttcttat
     4441 gatttatgat ttttattatt aaataagtta taaaaaaaat aagtgtatac aaattttaaa
     4501 gtgactctta ggttttaaaa cgaaaattct tattcttgag taactctttc ctgtaggtca
     4561 ggttgctttc tcaggtatag catgaggtcg ctcttattga ccacacctcc cttaaccaga
     4621 ttcgaaaagc ggcaagaaat gatggtaaat gaaataggaa atcaaggagc atgaaggcaa
     4681 aagacaaata taagggtcga acgaaaaata aagtgaaaag tgttgatatg atgtatttgg
     4741 ctttgcggcg ccgaaaaaac gagtttacgc aattgcacaa tcatgctgac tctgtggcgg
     4801 acccgcgctc ttgccggccc ggcgataacg ctgggcgtga ggctgtgccc ggcggagttt
     4861 tttgcgcctg cattttccaa ggtttaccct gcgctaaggg gcgagattgg agaagcaata
     4921 agaatgccgg ttggggttgc gatgatgacg accacgacaa ctggtgtcat tatttaagtt
     4981 gccgaaagaa cctgagtgca tttgcaacat gagtatacta gaagaatgag ccaagacttg
     5041 cgagacgcga gtttgccggt ggtgcgaaca atagagcgac catgaccttg aaggtgagac
     5101 gcgcataacc gctagagtac tttgaagagg aaacagcaat agggttgcta ccagtataaa
     5161 tagacaggta catacaacac tggaaatggt tgtctgtttg agtacgcttt caattcattt
     5221 gggtgtgcac tttattatgt tacaatatgg aagggaactt tacacttctc ctatgcacat
     5281 atattaatta aagtccaatg ctagtagaga aggggggtaa cacccctccg cgctcttttc
     5341 cgattttttt ctaaaccgtg gaatatttcg gatatccttt tgttgtttcc gggtgtacaa
     5401 tatggacttc ctcttttctg gcaaccaaac ccatacatcg ggattcctat aataccttcg
     5461 ttggtctccc taacatgtag gtggcggagg ggagatatac aatagaacag ataccagaca
     5521 agacataatg ggctaaacaa gactacacca attacactgc ctcattgatg gtggtacata
     5581 acgaactaat actgtagccc tagacttgat agccatcatc atatcgaagt ttcactaccc
     5641 tttttccatt tgccatctat tgaagtaata ataggcgcat gcaacttctt ttcttttttt
     5701 ttcttttctc tctcccccgt tgttgtctca ccatatccgc aatgacaaaa aaatgatgga
     5761 agacactaaa ggaaaaaatt aacgacaaag acagcaccaa cagatgtcgt tgttccagag
     5821 ctgatgaggg gtatctcgaa gcacacgaaa ctttttcctt ccttcattca cgcacactac
     5881 tctctaatga gcaacggtat acggccttcc ttccagttac ttgaatttga aataaaaaaa
     5941 agtttgctgt cttgctatca agtataaata gacctgcaat tattaatctt ttgtttcctc
     6001 gtcattgttc tcgttccctt tcttccttgt ttctttttct gcacaatatt tcaagctata
     6061 ccaagcatac aatcaactat ctcatataca ggattctaga actagtggat ctacaaatgt
     6121 cacctgatga gtccgtgagg acgaaacgag taagctcgtc gtgacataaa cattcgactc
     6181 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     6241 ggcaccgagt cggtgctttt ggccggcatg gtcccagcct cctcgctggc gccggctggg
     6301 caacatgctt cggcatggcg aatgggactg ataccgtcga cctcgagtca attagttatg
     6361 tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga
     6421 caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta
     6481 tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata
     6541 ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg
//
