LOCUS       sequence-449555-        3847 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast genome editing, Insertion of 20 bp oligo into sgRNA gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3847)
  AUTHORS   Kiyokawa K, Sakuma T, Moriguchi K, Sugiyama M, Akao T, Yamamoto T, 
            Suzuki K
  TITLE     Conversion of polyploid and alloploid Saccharomyces sensu stricto 
            strains to leu2 mutants by genome DNA editing.
  JOURNAL   Appl Microbiol Biotechnol. 2024 Jul 12;108(1):416. doi: 
            10.1007/s00253-024-13242-y.
  PUBMED    38995331
REFERENCE   2  (bases 1 to 3847)
  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: "Appl 
            Microbiol Biotechnol."; date: "2024-07-12"; volume: "108(1):416. 
            doi"; pages: " 10.1007/s00253-024-13242-y"
COMMENT     Sequence Label: pBSsgRNA
FEATURES             Location/Qualifiers
     source          1..3847
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     680..696
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     680..696
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    770..791
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(806..811,812..829,830..836)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    844..860
                     /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)."
     primer_bind     868..884
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        905..923
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        954..1222
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     terminator      1304..1323
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      complement(1441..1628)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1549..1573
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(1662..1680)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1701..1717)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1725..1741
                     /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(1749..1755,1756..1773,1774..1779))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1794..1815
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1932..1949)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2103..2691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2183..2202)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2862..3653,3654..3722))
                     /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     3485..3504
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3723..3827)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattggagct
      661 ccaccgcggt ggcggccgct ctagaactag tggatccccc gggctgcagg aattcgatct
      721 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
      781 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
      841 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
      901 gcgcaattaa ccctcactaa agggaacaaa agctggagct ccaccgcgga caatctttga
      961 aaagataatg tatgattatg ctttcactca tatttataca gaaacttgat gttttctttc
     1021 gagtatatac aaggtgatta catgtacgtt tgaagtacaa ctctagattt tgtagtgccc
     1081 tcttgggcta gcggtaaagg tgcgcatttt ttcacaccct acaatgttct gttcaaaaga
     1141 ttttggtcaa acgctgtaga agtgaaagtt ggtgcgcatg tttcggcgtt cgaaacttct
     1201 ccgcagtgaa agataaatga tcagaatcga tgcatttaaa tagcaagtta aaataaggct
     1261 agtccgttat caacttgaaa aagtggcacc gagtcggtgg tgcttttttt gttttttatg
     1321 tcttcgagtc atgtaattag ttataagcat gtgagctaag acactgtaat tgccaatcta
     1381 aacgatacca cggccgctct agagaaatgg ggagcgattt gcaggcattt gctcggcatg
     1441 ccggtagagg tgtggtcaat aagagcgacc tcatgctata cctgagaaag caacctgacc
     1501 tacaggaaag agttactcaa gaataagaat tttcgtttta aaacctaaga gtcactttaa
     1561 aatttgtata cacttatttt ttttataact tatttaataa taaaaatcat aaatcataag
     1621 aaattcgcct cgaggggggg cccggtaccc agcttttgtt ccctttagtg agggttaatt
     1681 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     1741 attccacaca acatacgagc cgggagcata aagtgtaaag cctggggtgc ctaatgagtg
     1801 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1861 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1921 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1981 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2041 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2101 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2161 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2221 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2281 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2341 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2401 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2461 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2521 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2581 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2641 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2701 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2761 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2821 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2881 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2941 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3001 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3061 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3121 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3181 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3241 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3301 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3361 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3421 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3481 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3541 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3601 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3661 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3721 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3781 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3841 aaagtgc
//
