LOCUS       sequence-77329-e        5839 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Same as pRS316-RGR-GFP except for the HDV ribozyme region which has 
            a 15 bp deletion at its 3' end..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5839)
  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 5839)
  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-mHDV
FEATURES             Location/Qualifiers
     source          1..5839
                     /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"
     terminator      2812..2999
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2867..2891)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3019..3035)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3020..3036)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(3065..3083)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3104..3120)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3104..3120)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3117..3139)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3128..3144
                     /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(3152..3158,3159..3176,3177..3182))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3335..3352)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3506..4094)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4265..5056,5057..5125))
                     /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     4888..4907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5126..5230)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    5267..5770
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     5812..5830
                     /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 ttcggagctt tggacttctt cgccagaggt
     2701 ttggtcaagt ctccaatcaa ggttgtcggc ttgtctacct tgccagaaat ttacgaaaag
     2761 atggaaaagg gtcaaatcgt tggtagatac gttgttgaca cttctaaata agcgaatttc
     2821 ttatgattta tgatttttat tattaaataa gttataaaaa aaataagtgt atacaaattt
     2881 taaagtgact cttaggtttt aaaacgaaaa ttcttgttct tgagtaactc tttcctgtag
     2941 gtcaggttgc tttctcaggt atagcatgag gtcgctctta ttgaccacac ctctaccggc
     3001 atgcgatatc aagcttatcg ataccgtcga cctcgagggg gggcccggta cccagctttt
     3061 gttcccttta gtgagggtta attccgagct tggcgtaatc atggtcatag ctgtttcctg
     3121 tgtgaaattg ttatccgctc acaattccac acaacatagg agccggaagc ataaagtgta
     3181 aagcctgggg tgcctaatga gtgaggtaac tcacattaat tgcgttgcgc tcactgcccg
     3241 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3301 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3361 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3421 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3481 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctcgg cccccctgac gagcatcaca
     3541 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3601 tcccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3661 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     3721 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3781 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3841 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3901 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     3961 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4021 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4081 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4141 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4201 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4261 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4321 ccatagttgc ctgactgccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4381 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4441 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4501 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4561 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4621 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgaaaaa
     4681 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4741 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     4801 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4861 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4921 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4981 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5041 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5101 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5161 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5221 gggttccgcg cacatttccc cgaaaagtgc cacctgggtc cttttcatca cgtgctataa
     5281 aaataattat aatttaaatt ttttaatata aatatataaa ttaaaaatag aaagtaaaaa
     5341 aagaaattaa agaaaaaata gtttttgttt tccgaagatg taaaagactc tagggggatc
     5401 gccaacaaat actacctttt atcttgctct tcctgctctc aggtattaat gccgaattgt
     5461 ttcatcttgt ctgtgtagaa gaccacacac gaaaatcctg tgattttaca ttttacttat
     5521 cgttaatcga atgtatatct atttaatctg cttttcttgt ctaataaata tatatgtaaa
     5581 gtacgctttt tgttgaaatt ttttaaacct ttgtttattt ttttttcttc attccgtaac
     5641 tcttctacct tctttattta ctttctaaaa tccaaataca aaacataaaa ataaataaac
     5701 acagagtaaa ttcccaaatt attccatcat taaaagatac gaggcgcgtg taagttacag
     5761 gcaagcgatc cgtcctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     5821 atcacgaggc cctttcgtc
//
