LOCUS       sequence-77328-e        5841 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Same as pRS316-RGR-GFP except for the HH ribozyme region which has a
            13 bp deletion at its 5' end..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5841)
  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 5841)
  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-mHH
FEATURES             Location/Qualifiers
     source          1..5841
                     /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"
     misc_RNA        2530..2605
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           2610..2677
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      2814..3001
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2869..2893)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3021..3037)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3022..3038)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(3067..3085)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3106..3122)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3106..3122)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3119..3141)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3130..3146
                     /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(3154..3160,3161..3178,3179..3184))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3337..3354)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3508..4096)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4267..5058,5059..5127))
                     /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     4890..4909
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5128..5232)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    5269..5772
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     5814..5832
                     /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 caatcaactg tccgtgagga cgaaacgagt aagctcgtcc gtgctgaagt
     2521 caagtttgag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
     2581 tgaaaaagtg gcaccgagtc ggtgcttttg gccggcatgg tcccagcctc ctcgctggcg
     2641 ccggctgggc aacatgcttc ggcatggcga atgggacagc tttggacttc ttcgccagag
     2701 gtttggtcaa gtctccaatc aaggttgtcg gcttgtctac cttgccagaa atttacgaaa
     2761 agatggaaaa gggtcaaatc gttggtagat acgttgttga cacttctaaa taagcgaatt
     2821 tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat
     2881 tttaaagtga ctcttaggtt ttaaaacgaa aattcttgtt cttgagtaac tctttcctgt
     2941 aggtcaggtt gctttctcag gtatagcatg aggtcgctct tattgaccac acctctaccg
     3001 gcatgcgata tcaagcttat cgataccgtc gacctcgagg gggggcccgg tacccagctt
     3061 ttgttccctt tagtgagggt taattccgag cttggcgtaa tcatggtcat agctgtttcc
     3121 tgtgtgaaat tgttatccgc tcacaattcc acacaacata ggagccggaa gcataaagtg
     3181 taaagcctgg ggtgcctaat gagtgaggta actcacatta attgcgttgc gctcactgcc
     3241 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3301 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3361 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3421 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3481 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc ggcccccctg acgagcatca
     3541 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3601 gttcccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3661 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta
     3721 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3781 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3841 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3901 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     3961 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4021 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4081 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4141 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4201 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4261 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4321 atccatagtt gcctgactgc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4381 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4441 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4501 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4561 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4621 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgaaa
     4681 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4741 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4801 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4861 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4921 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4981 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5041 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5101 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5161 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5221 aggggttccg cgcacatttc cccgaaaagt gccacctggg tccttttcat cacgtgctat
     5281 aaaaataatt ataatttaaa ttttttaata taaatatata aattaaaaat agaaagtaaa
     5341 aaaagaaatt aaagaaaaaa tagtttttgt tttccgaaga tgtaaaagac tctaggggga
     5401 tcgccaacaa atactacctt ttatcttgct cttcctgctc tcaggtatta atgccgaatt
     5461 gtttcatctt gtctgtgtag aagaccacac acgaaaatcc tgtgatttta cattttactt
     5521 atcgttaatc gaatgtatat ctatttaatc tgcttttctt gtctaataaa tatatatgta
     5581 aagtacgctt tttgttgaaa ttttttaaac ctttgtttat ttttttttct tcattccgta
     5641 actcttctac cttctttatt tactttctaa aatccaaata caaaacataa aaataaataa
     5701 acacagagta aattcccaaa ttattccatc attaaaagat acgaggcgcg tgtaagttac
     5761 aggcaagcga tccgtcctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5821 gtatcacgag gccctttcgt c
//
