LOCUS       sequence-99568-e        6067 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  crRNA expression plasmid on pRS423 backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6067)
  AUTHORS   Bao Z, Xiao H, Liang J, Zhang L, Xiong X, Sun N, Si T, Zhao H
  TITLE     Homology-Integrated CRISPR-Cas (HI-CRISPR) System for One-Step 
            Multigene Disruption in Saccharomyces cerevisiae.
  JOURNAL   ACS Synth Biol. 2014 Sep 19.
  PUBMED    25207793
REFERENCE   2  (bases 1 to 6067)
  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: "ACS Synth 
            Biol. 2014 Sep 19."
COMMENT     Sequence Label: pCR
FEATURES             Location/Qualifiers
     source          1..6067
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        317..504
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             505..1164
                     /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"
     primer_bind     complement(1561..1582)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1658..1926
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     CDS             complement(1969..2265)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSG"
     primer_bind     2115..2137
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2129..2146
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2130..2146
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2156..2175
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2156..2174
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2222..2242)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2222..2240)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2261..2277)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2285..2301
                     /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(2309..2315,2316..2333,2334..2339))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2354..2375
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     terminator      2448..2467
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(2505..2521)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2529..2545
                     /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(2553..2559,2560..2577,2578..2583))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2598..2619
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2736..2753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2907..3495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2987..3006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3666..4457,4458..4526))
                     /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     4289..4308
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4527..4631)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4658..6000
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5621..5668)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     6040..6058
                     /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 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt
      241 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta
      301 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat
      361 tttttttttt ccacctagcg gatgactctt tttttttctt agcgattggc attatcacat
      421 aatgaattat acattatata aagtaatgtg atttcttcga agaatatact aaaaaatgag
      481 caggcaagat aaacgaaggc aaagatgaca gagcagaaag ccctagtaaa gcgtattaca
      541 aatgaaacca agattcagat tgcgatctct ttaaagggtg gtcccctagc gatagagcac
      601 tcgatcttcc cagaaaaaga ggcagaagca gtagcagaac aggccacaca atcgcaagtg
      661 attaacgtcc acacaggtat agggtttctg gaccatatga tacatgctct ggccaagcat
      721 tccggctggt cgctaatcgt tgagtgcatt ggtgacttac acatagacga ccatcacacc
      781 actgaagact gcgggattgc tctcggtcaa gcttttaaag aggccctact ggcgcgtgga
      841 gtaaaaaggt ttggatcagg atttgcgcct ttggatgagg cactttccag agcggtggta
      901 gatctttcga acaggccgta cgcagttgtc gaacttggtt tgcaaaggga gaaagtagga
      961 gatctctctt gcgagatgat cccgcatttt cttgaaagct ttgcagaggc tagcagaatt
     1021 accctccacg ttgattgtct gcgaggcaag aatgatcatc accgtagtga gagtgcgttc
     1081 aaggctcttg cggttgccat aagagaagcc acctcgccca atggtaccaa cgatgttccc
     1141 tccaccaaag gtgttcttat gtagtgacac cgattattta aagctgcagc atacgatata
     1201 tatacatgtg tatatatgta tacctatgaa tgtcagtaag tatgtatacg aacagtatga
     1261 tactgaagat gacaaggtaa tgcatcattc tatacgtgtc attctgaacg aggcgcgctt
     1321 tccttttttc tttttgcttt ttcttttttt ttctcttgaa ctcgacggat ctatgcggtg
     1381 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat
     1441 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     1501 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     1561 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     1621 accgtctatc agggcgatgg cccactacgt gctcgagtct ttgaaaagat aatgtatgat
     1681 tatgctttca ctcatattta tacagaaact tgatgttttc tttcgagtat atacaaggtg
     1741 attacatgta cgtttgaagt acaactctag attttgtagt gccctcttgg gctagcggta
     1801 aaggtgcgca ttttttcaca ccctacaatg ttctgttcaa aagattttgg tcaaacgctg
     1861 tagaagtgaa agttggtgcg catgtttcgg cgttcgaaac ttctccgcag tgaaagataa
     1921 atgatcgggt tttagagcta tgctgttttg aatggtccca aagagacctc agccgctaca
     1981 gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     2041 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     2101 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata
     2161 cgactcacta tagggcgaat tgggtaccgg gccccccctc gaggtcctcc agcttttgtt
     2221 ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt
     2281 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     2341 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     2401 tccaccggtg gtctctagag ctatgctgtt ttgaatggtc ccaaaacttt ttttgttttt
     2461 tatgtctgag ctccctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct
     2521 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     2581 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     2641 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     2701 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2761 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2821 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2881 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2941 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3001 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     3061 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     3121 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     3181 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     3241 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     3301 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     3361 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     3421 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3481 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3541 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3601 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3661 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3721 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3781 ggccccagtg ctgcaatgat accgcggctc ccacgctcac cggctccaga tttatcagca
     3841 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3901 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3961 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     4021 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     4081 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     4141 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     4201 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     4261 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     4321 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     4381 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     4441 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     4501 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     4561 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4621 ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac gaagcatctg tgcttcattt
     4681 tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat
     4741 ttttacagaa cagaaatgca acgcgaaagc gctattttac caacgaagaa tctgtgcttc
     4801 atttttgtaa aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc
     4861 tgcattttta cagaacagaa atgcaacgcg agagcgctat tttaccaaca aagaatctat
     4921 acttcttttt tgttctacaa aaatgcatcc cgagagcgct atttttctaa caaagcatct
     4981 tagattactt tttttctcct ttgtgcgctc tataatgcag tctcttgata actttttgca
     5041 ctgtaggtcc gttaaggtta gaagaaggct actttggtgt ctattttctc ttccataaaa
     5101 aaagcctgac tccacttccc gcgtttactg attactagcg aagctgcggg tgcatttttt
     5161 caagataaag gcatccccga ttatattcta taccgatgtg gattgcgcat actttgtgaa
     5221 cagaaagtga tagcgttgat gattcttcat tggtcagaaa attatgaacg gtttcttcta
     5281 ttttgtctct atatactacg tataggaaat gtttacattt tcgtattgtt ttcgattcac
     5341 tctatgaata gttcttacta caattttttt gtctaaagag taatactaga gataaacata
     5401 aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga tgggtaggtt
     5461 atatagggat atagcacaga gatatatagc aaagagatac ttttgagcaa tgtttgtgga
     5521 agcggtattc gcaatatttt agtagctcgt tacagtccgg tgcgtttttg gttttttgaa
     5581 agtgcgtctt cagagcgctt ttggttttca aaagcgctct gaagttccta tactttctag
     5641 agaataggaa cttcggaata ggaacttcaa agcgtttccg aaaacgagcg cttccgaaaa
     5701 tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc gcacctatat ctgcgtgttg
     5761 cctgtatata tatatacatg agaagaacgg catagtgcgt gtttatgctt aaatgcgtac
     5821 ttatatgcgt ctatttatgt aggatgaaag gtagtctagt acctcctgtg atattatccc
     5881 attccatgcg gggtatcgta tgcttccttc agcactaccc tttagctgtt ctatatgctg
     5941 ccactcctca attggattag tctcatcctt caatgctatc atttcctttg atattggatc
     6001 atctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     6061 tttcgtc
//
