LOCUS       sequence-77326-e        5854 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express self-cleaving-ribozyme-flanked sgRNA cassette (RGR) 
            targeting GFP for CRISPR systems in yeast driven by ADH1 promoter. 
            RGR has a HH ribozyme at its 5', and an HDV ribozyme at its 3'..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5854)
  AUTHORS   Gao Y, Zhao Y
  TITLE     Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro 
            and in vivo for CRISPR-mediated genome editing.
  JOURNAL   J Integr Plant Biol. 2013 Dec 30. doi: 10.1111/jipb.12152.
  PUBMED    24373158
REFERENCE   2  (bases 1 to 5854)
  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: "J Integr 
            Plant Biol. 2013 Dec 30. doi: 10.1111/jipb.12152."
COMMENT     Sequence Label: pRS316-RGR-GFP
FEATURES             Location/Qualifiers
     source          1..5854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        190..410
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             411..1214
                     /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
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1343..1798)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1480..1501)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1692..1711
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1928..1950
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1942..1959
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1943..1959
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1966..1985
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1966..1984
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        2078..2479
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     ncRNA           2486..2522
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        2543..2618
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           2623..2690
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      2827..3014
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2882..2906)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3034..3050)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3035..3051)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(3080..3098)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3119..3135)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3119..3135)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3132..3154)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3143..3159
                     /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(3167..3173,3174..3191,3192..3197))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3350..3367)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3521..4109)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4280..5071,5072..5140))
                     /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     4903..4922
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5141..5245)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    5282..5785
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     5827..5845
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accacgcttt tcaattcaat tcatcatttt ttttttattc ttttttttga tttcggtttc
      241 tttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca
      301 gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat
      361 tcttaaccca actgcacaga acaaaaacct gcaggaaacg aagataaatc atgtcgaaag
      421 ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata
      481 tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat
      541 tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata
      601 tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt
      661 acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc
      721 agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg
      781 tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac
      841 ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat
      901 atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg
      961 ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg
     1021 tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg gatgatgtgg
     1081 tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg
     1141 ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg
     1201 gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac tcacaaatta
     1261 gagcttcaat ttaattatat cagttattac cctgcggtgt gaaataccgc acagatgcgt
     1321 aaggagaaaa taccgcatca ggaaattgta aacgttaata ttttgttaaa attcgcgtta
     1381 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     1441 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     1501 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     1561 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     1621 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     1681 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     1741 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg
     1801 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     1861 ctattacgcc agctggcgaa ggggggatgt gctgcaaggc gattaagttg ggtaacgcca
     1921 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta
     1981 tagggcgaat tggagctcca ccgcggtggc ggccgctcta gaactagtgg gagctgcatg
     2041 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcaacttct tttctttttt
     2101 tttcttttct ctctcccccg ttgttgtctc accatatccg caatgacaaa aaaatgatgg
     2161 aagacactaa aggaaaaaat taacgacaaa gacagcacca acagatgtcg ttgttccaga
     2221 gctgatgagg ggtatctcga agcacacgaa actttttcct tccttcattc acgcacacta
     2281 ctctctaatg agcaacggta tacggccttc cttccagtta cttgaatttg aaataaaaaa
     2341 aagtttgctg tcttgctatc aagtataaat agacctgcaa ttattaatct tttgtttcct
     2401 cgtcattgtt ctcgttccct ttcttccttg tttctttttc tgcacaatat ttcaagctat
     2461 accaagcata caatcaacta gcacgctgat gagtccgtga ggacgaaacg agtaagctcg
     2521 tccgtgctga agtcaagttt gagttttaga gctagaaata gcaagttaaa ataaggctag
     2581 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttggccggca tggtcccagc
     2641 ctcctcgctg gcgccggctg ggcaacatgc ttcggcatgg cgaatgggac agctttggac
     2701 ttcttcgcca gaggtttggt caagtctcca atcaaggttg tcggcttgtc taccttgcca
     2761 gaaatttacg aaaagatgga aaagggtcaa atcgttggta gatacgttgt tgacacttct
     2821 aaataagcga atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata
     2881 agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt gttcttgagt
     2941 aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc tcttattgac
     3001 cacacctcta ccggcatgcg atatcaagct tatcgatacc gtcgacctcg agggggggcc
     3061 cggtacccag cttttgttcc ctttagtgag ggttaattcc gagcttggcg taatcatggt
     3121 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac ataggagccg
     3181 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag gtaactcaca ttaattgcgt
     3241 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3301 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3361 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3421 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3481 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctcggccccc
     3541 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3601 aaagatacca ggcgttcccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3661 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct
     3721 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3781 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3841 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3901 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3961 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4021 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4081 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4141 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4201 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4261 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4321 gtctatttcg ttcatccata gttgcctgac tgcccgtcgt gtagataact acgatacggg
     4381 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4441 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4501 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4561 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4621 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4681 ccatgttgtg aaaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4741 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4801 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4861 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4921 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4981 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5041 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5101 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5161 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5221 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gggtcctttt
     5281 catcacgtgc tataaaaata attataattt aaatttttta atataaatat ataaattaaa
     5341 aatagaaagt aaaaaaagaa attaaagaaa aaatagtttt tgttttccga agatgtaaaa
     5401 gactctaggg ggatcgccaa caaatactac cttttatctt gctcttcctg ctctcaggta
     5461 ttaatgccga attgtttcat cttgtctgtg tagaagacca cacacgaaaa tcctgtgatt
     5521 ttacatttta cttatcgtta atcgaatgta tatctattta atctgctttt cttgtctaat
     5581 aaatatatat gtaaagtacg ctttttgttg aaatttttta aacctttgtt tatttttttt
     5641 tcttcattcc gtaactcttc taccttcttt atttactttc taaaatccaa atacaaaaca
     5701 taaaaataaa taaacacaga gtaaattccc aaattattcc atcattaaaa gatacgaggc
     5761 gcgtgtaagt tacaggcaag cgatccgtcc taagaaacca ttattatcat gacattaacc
     5821 tataaaaata ggcgtatcac gaggcccttt cgtc
//
