LOCUS       sequence-77330-e        5826 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, and 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 5826)
  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 5826)
  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-mm
FEATURES             Location/Qualifiers
     source          1..5826
                     /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"
     terminator      2799..2986
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2854..2878)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3006..3022)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3007..3023)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(3052..3070)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3091..3107)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3091..3107)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3104..3126)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3115..3131
                     /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(3139..3145,3146..3163,3164..3169))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3322..3339)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3493..4081)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4252..5043,5044..5112))
                     /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     4875..4894
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5113..5217)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    5254..5757
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     5799..5817
                     /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 ggagctttgg acttcttcgc cagaggtttg gtcaagtctc
     2701 caatcaaggt tgtcggcttg tctaccttgc cagaaattta cgaaaagatg gaaaagggtc
     2761 aaatcgttgg tagatacgtt gttgacactt ctaaataagc gaatttctta tgatttatga
     2821 tttttattat taaataagtt ataaaaaaaa taagtgtata caaattttaa agtgactctt
     2881 aggttttaaa acgaaaattc ttgttcttga gtaactcttt cctgtaggtc aggttgcttt
     2941 ctcaggtata gcatgaggtc gctcttattg accacacctc taccggcatg cgatatcaag
     3001 cttatcgata ccgtcgacct cgaggggggg cccggtaccc agcttttgtt ccctttagtg
     3061 agggttaatt ccgagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3121 tccgctcaca attccacaca acataggagc cggaagcata aagtgtaaag cctggggtgc
     3181 ctaatgagtg aggtaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3241 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3301 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     3361 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     3421 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3481 gttgctggcg tttttccata ggctcggccc ccctgacgag catcacaaaa atcgacgctc
     3541 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgttcc cccctggaag
     3601 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3661 cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta
     3721 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3781 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3841 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3901 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     3961 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4021 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4081 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4141 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4201 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4261 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4321 actgcccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     4381 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     4441 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4501 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4561 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4621 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgaaaaaaag cggttagctc
     4681 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     4741 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     4801 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     4861 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4921 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     4981 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5041 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5101 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5161 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5221 atttccccga aaagtgccac ctgggtcctt ttcatcacgt gctataaaaa taattataat
     5281 ttaaattttt taatataaat atataaatta aaaatagaaa gtaaaaaaag aaattaaaga
     5341 aaaaatagtt tttgttttcc gaagatgtaa aagactctag ggggatcgcc aacaaatact
     5401 accttttatc ttgctcttcc tgctctcagg tattaatgcc gaattgtttc atcttgtctg
     5461 tgtagaagac cacacacgaa aatcctgtga ttttacattt tacttatcgt taatcgaatg
     5521 tatatctatt taatctgctt ttcttgtcta ataaatatat atgtaaagta cgctttttgt
     5581 tgaaattttt taaacctttg tttatttttt tttcttcatt ccgtaactct tctaccttct
     5641 ttatttactt tctaaaatcc aaatacaaaa cataaaaata aataaacaca gagtaaattc
     5701 ccaaattatt ccatcattaa aagatacgag gcgcgtgtaa gttacaggca agcgatccgt
     5761 cctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     5821 ttcgtc
//
