LOCUS       sequence-199274-        6058 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli/S. cerevisiae amdS shuttle vector expressing a ribozyme 
            flanked g-RNA for Cas9 editing targeting the gene SeILV6 in S. 
            pastorianus (HH-gRNASeILV6-HDV).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6058)
  AUTHORS   de Vries ARG, de Groot PA, van den Broek M, Daran JG
  TITLE     CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces 
            pastorianus.
  JOURNAL   Microb Cell Fact. 2017 Dec 5;16(1):222. doi: 
            10.1186/s12934-017-0835-1.
  PUBMED    29207996
REFERENCE   2  (bases 1 to 6058)
  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: "Microb Cell
            Fact."; date: "2017-12-5"; volume: "16(1):222. doi"; pages: " 
            10.1186/s12934-017-0835-1"
COMMENT     Sequence Label: pUDP010
FEATURES             Location/Qualifiers
     source          1..6058
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2868..3534
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3404..3427
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     ncRNA           3541..3577
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        3598..3673
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           3678..3745
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     primer_bind     complement(3787..3805)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(join(4239..5030,5031..5099))
                     /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     4862..4881
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5100..5204)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5307..5895
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5796..5815
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga
       61 tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata
      121 cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag
      181 cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga
      241 aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc
      301 ttgctaggat acagttctca catcacatcc gaacataaac aaggatccat gccacaatct
      361 tgggaagaat tggctgctga caagagagct agattggcta agactatccc agacgaatgg
      421 aaggttcaaa ctttgccagc tgaagactct gttatcgact tcccaaagaa gtctggtatc
      481 ttgtctgaag ctgaattgaa gatcactgaa gcttctgctg ctgacttggt ttctaagttg
      541 gctgctggtg aattgacttc tgttgaagtt actttggctt tctgtaagag agctgctatc
      601 gctcaacaat tgactaactg tgctcacgaa ttcttcccag acgctgcttt ggctcaagct
      661 agagaattgg acgaatacta cgctaagcac aaaagacctg ttggtccatt gcacggtttg
      721 ccaatctctt tgaaggacca attgagagtt aagggttacg aaacttctat gggttacatc
      781 tcttggttga acaagtacga cgaaggtgac tctgttttga ctactatgtt gagaaaggct
      841 ggtgctgttt tctacgttaa gacttctgtt ccacaaactt tgatggtttg tgaaactgtt
      901 aacaacatca tcggtagaac tgttaaccca agaaacaaga actggtcttg tggtggttct
      961 tctggtggtg aaggtgctat cgttggtatc agaggtggtg ttatcggtgt tggtactgac
     1021 atcggtggtt ctatcagagt tccagctgct ttcaacttct tgtacggttt aagaccatct
     1081 cacggtagat tgccatacgc taagatggct aactctatgg aaggtcaaga aactgtacac
     1141 tctgttgttg gtccaatcac tcactctgtt gaagacttga gattgttcac taagtctgtt
     1201 ttgggtcaag aaccatggaa gtacgactct aaggttatcc caatgccatg gagacaatct
     1261 gaatctgaca tcatcgcttc taagatcaag aacggtggtt tgaacatcgg ttactacaac
     1321 ttcgacggta acgttttgcc acacccacca atcttgagag gtgttgaaac tactgttgct
     1381 gctttggcta aggctggtca cactgttact ccatggactc catacaagca cgacttcggt
     1441 cacgacttga tctctcacat ctacgctgct gacggttctg ctgacgttat gagagacatc
     1501 tctgcttctg gtgaaccagc tatcccaaac atcaaggact tgttgaaccc aaacatcaag
     1561 gctgttaaca tgaacgaatt gtgggacact cacttgcaaa agtggaacta ccaaatggaa
     1621 tacttggaaa agtggagaga agctgaagaa aaggctggta aggaattgga cgctatcatc
     1681 gctccaatca ctccaactgc tgctgttaga cacgaccaat tcagatacta cggttacgct
     1741 tctgttatca acttgttgga cttcacttct gttgttgttc cagttacttt cgctgacaag
     1801 aacatcgaca agaagaacga atctttcaag gctgtttctg aattggacgc tttggttcaa
     1861 gaagaatacg acccagaagc ttaccacggt gctccagttg ctgttcaagt tatcggtaga
     1921 agattgtctg aagaaagaac tttggctatc gctgaagaag ttggtaagtt gttgggtaac
     1981 gttgttactc cataactcga ggacaataaa aagattcttg ttttcaagaa cttgtcattt
     2041 gtatagtttt tttatattgt agttgttcta ttttaatcaa atgttagcgt gatttatatt
     2101 tttttcgcct cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat
     2161 gcgtcaatcg tatgtgaatg ctggtcgcta tactggagct ctcgagaacc ctcacctttc
     2221 gagaggacga tgcccgtgtc taaatgattc gaccagccta agaatgttca actcaacatc
     2281 tttggataat atcagaatga gaaagaacag atacgcagta cgttttttgg tgagctcttt
     2341 gcacttcttt agttctttcc atcaatatca gttttttaaa cttttaggac taaaagtgat
     2401 gtttaacttc aaaatgttta aaattttgtt cttcccgacg ttcattaaga atactaatac
     2461 actttaataa ttattttaat attttgttct aaataatgac ttttaattaa aaaagataaa
     2521 atataaaaac atcataataa ctcaccagag gttaagaaca aaaaaacaaa ttagatatct
     2581 gctaatccaa tatagttaaa tcaatctttc cttggtataa tgggtatatt acatatattt
     2641 caaggaccga cactcctacc aaatatctaa aatttaccat attaacataa catgtatata
     2701 aacgtcaaat cataatcagc actaacgtct cacggatcgt atatgccgta gcgacaatct
     2761 aagaactatg cgaggacacg ctagggccaa ggcaaaaaga ttccttgatt acgtaaggga
     2821 gttagaatca ttttgaataa aaaacacgct ttttcagttc gagtttatca ttatcaatac
     2881 tgccatttca aagaatacgt aaataattaa tagtagtgat tttcctaact ttatttagtc
     2941 aaaaaattag ccttttaatt ctgctgtaac ccgtacatgc ccaaaatagg gggcgggtta
     3001 cacagaatat ataacatcgt aggtgtctgg gtgaacagtt tattcctggc atccactaaa
     3061 tataatggag cccgcttttt aagctggcat ccagaaaaaa aaagaatccc agcaccaaaa
     3121 tattgttttc ttcaccaacc atcagttcat aggtccattc tcttagcgca actacagaga
     3181 acaggggcac aaacaggcaa aaaacgggca caacctcaat ggagtgatgc aacctgcctg
     3241 gagtaaatga tgacacaagg caattgaccc acgcatgtat ctatctcatt ttcttacacc
     3301 ttctattacc ttctgctctc tctgatttgg aaaaagctga aaaaaaaggt tgaaaccagt
     3361 tccctgaaat tattccccta cttgactaat aagtatataa agacggtagg tattgattgt
     3421 aattctgtaa atctatttct taaacttctt aaattctact tttatagtta gtcttttttt
     3481 tagttttaaa acaccaagaa cttagtttcg aataaacaca cataaacaaa caaacaattc
     3541 ctgatgagtc cgtgaggacg aaacgagtaa gctcgtcgaa ttgcttttct ctgatttgtt
     3601 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     3661 accgagtcgg tgcttttggc cggcatggtc ccagcctcct cgctggcgcc ggctgggcaa
     3721 catgcttcgg catggcgaat gggacacagg ccccttttcc tttgtcgata tcatgtaatt
     3781 agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg
     3841 agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag
     3901 aacgttattt atatttcaaa tttttctttt ttttctgtac aaacgcgtgt acgcatgtaa
     3961 cattatactg aaaaccttgc ttgagaaggt tttgggacaa gcttacgtct cacggatcgt
     4021 atatgccgta gcgacaatct aagaactatg cgaggacacg ctagcagctg tattcacgta
     4081 gacggatagg tatagccaga catcagcagc atacttcggg aaccgtaggc caccgagcgg
     4141 ccgcgattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4201 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4261 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4321 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcgg
     4381 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4441 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4501 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4561 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4621 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4681 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4741 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4801 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4861 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4921 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4981 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5041 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5101 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5161 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5221 gtgccacctg tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5281 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5341 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5401 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     5461 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5521 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5581 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5641 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5701 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5761 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5821 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5881 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5941 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtgcggac
     6001 tatatgtgaa ggcatggcta tggcacggca gacattccgc cagatcatca ataggcac
//
