LOCUS       sequence-441240-       10052 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning backbone for sgRNA in Kluyveromyces marxianus. Expresses 
            highly specific SlugCas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10052)
  TITLE     Wang lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 10052)
  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: 
            "Unpublished"
COMMENT     Sequence Label: LHZ1493
FEATURES             Location/Qualifiers
     source          1..10052
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2677..2693)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2677..2693)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2690..2712)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2701..2717
                     /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(2725..2731,2732..2749,2750..2755))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2770..2791
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2908..2925)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3079..3667)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3159..3178)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3838..4629,4630..4698))
                     /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     4461..4480
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4699..4803)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4871..4889
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4927..4949)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5049..5068
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5262..5284
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5276..5293
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5277..5293
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5305..5324
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5305..5323
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(5346..5365)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(5366..5441)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     terminator      complement(6067..6314)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     6280..6298
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(6327..6374)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(9558..9578)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(9603..10001)
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
ORIGIN
        1 tatttacctt ttaacctaca tattcatagt tggaagttat ccttctaagt acgtatacaa
       61 tattaattca acgtaaaaac aaaacttact gtaaatatgt gtaaaaaaaa tctattaaat
      121 tcatggcagt ttcaagaaaa gaaaactatt atggtctggt cacgtgtata caaattatta
      181 attttaaaac tatataattt attatttttt tattttgaag tttagagtaa ttttagtagt
      241 attttatatt ttaaataaat atgctttaaa tttttactta atattttatt atttttaaat
      301 acaacgtttt tatttaaaac aaaattataa gttaaaaagt tgttccgaaa gtaaaatata
      361 ttttatgggt tttacaaaaa taaattattt ttaatgtatt tttttaatta tatttttgta
      421 tgtaattata tccacaggta ttatgttgaa tttagctgtt ttagtttacc tgtgtggtac
      481 tatgattttt ttagaactct cctcttagaa ataggtggtg ttgcggttga cttttaacga
      541 tatatcattt tcaatttatt tattttaaag tgacatagag agattccttt taatttttta
      601 atttttattt tcaataattt taaaaatggg ggacttttaa attggaacaa aatgaaaaat
      661 atctgttata cgtgcaactg aattttactg accttaaagg actatctcga acttggttcg
      721 gaaatccttg aaatgattga tattttggtg gattttctct gattttcaaa caagtagtat
      781 tttatttaat atttattata ttttttacat ttttttatat ttttttattg tttggaaggt
      841 aaagcaacaa ttactttcaa aatatataaa tcaaactgaa atacttaata agagacaaat
      901 aacattcaag aatcaaatac tgggttatta atcaaaagat ctctctacat gcgcccaaat
      961 tcactattta aatttactat accactgaca gaatatatga acccagatta agtagccaga
     1021 ggtctcttcc actatattga gtatatagcc ttacatattt tctgcgcata atttactgat
     1081 gtaaaataaa caaaaatagt tagtttgtag ttatgaaaaa aggcttttgg aaaatgcgaa
     1141 atacgtgtta tttaaggtta atcaacaaaa cgcatatcca tagtggatag ttggacaaaa
     1201 cttcaatcga cccgggattt ccgagtacac tcgaacctct gcttgttctc ggagaactct
     1261 taagcggatc tgcctactct cttcaagtaa gcgtcccatc ccgcctttct gtatctctca
     1321 ccttccacta caggatctct tccctttgcg aaaagacctc taccaacaat aatgatgtct
     1381 gatccaccgg caacaacttc atccacagtt ctgtattgtt gtcccaaagc atcacctttg
     1441 tcatcaagac caacacctgg cgtcatgatc aaccaatcgt agccctcttc tcttccaccc
     1501 atatcgtttt gagcaataaa tccaataaca aagtccttat cactcttggc aatttccacg
     1561 gtcccacgag tgtattcacc gtgcgctaga gaccccttgg acgataactc ggcaagcatt
     1621 aacaaccctc taggttcttt tgtaacttcc tcggcacctt gcttcaaacc agcaacaatg
     1681 cccgcaccag tcacaccgtg tgcattggtg atatcagacc attcggcgat acggtataca
     1741 ccagacgtgt attgtaattt aacagtgttc ccaatgtcgg caaacttcct gtcttcaaat
     1801 atcaaaaact tgtgtttctc tgctaattgc ttcaacggca caatggtatt ctcaaagctg
     1861 aaatcctcca agatatctac atgtgtcttc aatagacaga tatatggacc caaaacctca
     1921 actaatctta acaactctgc tgttttacga acatcaagag aagcacataa gtttgacttc
     1981 ttctcttcca tcaagtttaa aagcttggca gcaactggac ttctatgagc agctgctctt
     2041 tccgagtaac tcttagtcga catcctcctt tgattagttt atggggctag ctgatgatac
     2101 ttcctcttgc tgttccagtt tacaaatgat ttaaacgaac ttttacactt ttacggtaga
     2161 atttcttgtt ggtgatgagc tcagattcaa tatgactgct taatcagtga aaattaatgg
     2221 aaacagccag acacacagcc tttgaaaaat gttaaaaacg atcacatata tgtagtcacg
     2281 tggttagtgc atagagtcag tcgatggtgg ttagttccct agccagagcc attcccctct
     2341 tgtttcgaaa tgcgatgact gaatggggaa gaagctctta gtatttctgt agtttttttt
     2401 gccgatttct attgttgcct gccccggact ttcttttacc ttttgtagct acaactatta
     2461 attataatgc ataatagatc tagaagcgta ttgtgatgag catccttgga aataagaaag
     2521 gaagtcaaag gtacagtaaa aaaagagccg tgacaggcac ctaatttcag ttttgattga
     2581 ggtataatga gctcagaaga ccaagatgct ctaaaagtaa agcagaatgg tctttccaat
     2641 cagaattcga ctagtgcaag cccgcgggta atcatggtca tagctgtttc ctgtgtgaaa
     2701 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     2761 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     2821 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     2881 tttgcgtatt gggcactctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     2941 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3001 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3061 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3121 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3181 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3241 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3301 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3361 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3421 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3481 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     3541 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3601 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3661 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3721 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3781 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     3841 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3901 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     3961 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4021 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4081 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4141 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4201 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4261 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4321 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4381 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4441 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4501 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4561 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4621 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4681 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4741 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     4801 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     4861 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     4921 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     4981 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
     5041 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     5101 gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa
     5161 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     5221 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     5281 aacgacggcc agtgcttaat taactaatac gactcactat agggcgaatt gggtaccgcg
     5341 gccgcagaca taaaaaacaa aaaaatctcg ccaacaagtt gacgagataa acacggcatt
     5401 ttgccttgtt ttagtagatt ctgtttccag agtactaaaa ctgaagagcc atggctcttc
     5461 ttgacactga cgggattcga acccgtgtca ctccctcgcc ttgttacttc tttttaggaa
     5521 gcaacttgtt aagtggaagg ggagtatcct aaaccactag actacagtgt cgagttcgtg
     5581 gtagaagaga aattgcggcc gcatctaaat tctctctttt tccttcaact tttattttct
     5641 tggtatgctt cttgtgcatt cgcctattaa tttatattag cgattcttat gacatcggcc
     5701 atcactgagt atagtgagta gtagtggttg atatttgaaa tctcttatag ataagttcct
     5761 ttacacatcg tgcagcactt gtttatgtga actattagta gctatattgg ctatatacag
     5821 agagaaagct ataactattt atatcataac tgtttgtata acttaacgac ccatgctttg
     5881 gttcaaagca gcgttttgct cttggaaacg ttgtccaaca cgttcactat gtttcaagaa
     5941 cttttctgag catttcatta tgcagctttg ctccttggaa gttagcttag cagaggtaaa
     6001 gtcgttgaca cagtcactga aacatctttc gaccaagttg gagtataagc gggcccggta
     6061 ccggccgcaa attaaagcct tcgagcgtcc caaaaccttc tcaagcaagg ttttcagtat
     6121 aatgttacat gcgtacacgc gtctgtacag aaaaaaaaga aaaatttgaa atataaataa
     6181 cgttcttaat actaacataa ctataaaaaa ataaataggg acctagactt caggttgtct
     6241 aactccttcc ttttcggtta gagcggatgt ggggggaggg cgtgaatgta agcgtgacat
     6301 aactaattac atgactcgag aagagacttt ttcttttttg cctggccggc ctttttcgtg
     6361 gccgccggcc ttttatttcc tcgcttgaac agcagctgag gttttgtgta ctgggtgttg
     6421 gtgaacacgt tgcccagcac gtcggttgtc agtttttcga tgctattcac ctttttgccg
     6481 atggtctttt tgattctagg ttcgcccttg atattgttca gctcgcagta ttctttgtat
     6541 ctgatgtcag gcagatccag ttcgatcatg tttctggtgt cgctattcac gccgatgatc
     6601 ttgtagatct cgccgtccag cttgatcaga tcattcttgt agaagctagc gataaacttg
     6661 gcgtttttgt cgatggcctt gcccagtttg agcttgtcgt acttctgttc gggtatgtag
     6721 tagtaattgt ctttcttcag cacgtccagg tagctgatgg tgatgaactt atatcccttg
     6781 tcggtcaggt agacgtcgaa tctataaggc ttgatgctca gcttcaccaa cttcttggtg
     6841 ctgctcttga actggtgggt cacatctagg tggctgccca gcttgtttcc aatgtacttc
     6901 aggcttttca cgatagggcc gttgtttttt ttgctgtatt tggtcaggta ctcgcctgtt
     6961 tcctcgtggt acttggccag aggattcttc tcattagcgt actgcttcat gatcacttcc
     7021 aacttctcga aggtccgcgg atcgtgttgg tacatgagga acttctcggg tgacttgtca
     7081 aactgcttct tcagggtggt attatctttg gcatagatat ctttgatggt ctgcacgatg
     7141 taggtggagt tgtccttctt ccgggtgctg tacagtgtat cgttgatcag ctgtctgtta
     7201 ggcttcttgt ccactctgtg gctgtacttg aagtttctga agtccttgat gtcctggacc
     7261 tgcttaggga tgatgaacat ttcgctgtag ttatcctcgc tatccacctg gatatccagc
     7321 tgcttgctct cgatctcagg tttttccagc acagagttca cggctttcag cttcttgttc
     7381 tctttgaaca ggaaatcggc attagcgata atcagggcgt cctcggcgtg gtgcttgtag
     7441 ccgtggttcc gttccttctt aaacttccac actttccgca ggtaatcggt gaagctgccg
     7501 ttgatcgttt tcaccttcac gttcatattg ttggcgctga agtaggcctt caggtagttt
     7561 gtcagctcgg ctgtggcgta ccgggtatcc accaggtttc tattgatgaa ctctttttgc
     7621 acctcgaact tgttgatgtc ccgttcttcc agcagatact ctttcttttt tttgctgatt
     7681 ctatcctggg acttgctcag gttcaggatg tgttgtttaa actggttgta agagagcttg
     7741 gacttgccgc tgttaaagta ctggtaaggg gtcaggttgg attttttgga gttctcgctc
     7801 tgcttcacga gcactttgtt gtggtaagag ttgtcgaagg acacagatct tggaatgatg
     7861 tggtccacct cgtagtggtt ggggttgttc agcaggtcct ccagagggat gctttccagt
     7921 gagtacaggc acttgccctc ctgctcgtcg tgcagtctaa tcttctcgac cagtctcttg
     7981 gcgttctggt tgccatactt gccgatgatt tcgttaattc tctttctggt gttctcgttc
     8041 tttttctgca tctcattgat gaacttctgc ttgtccttgc tattattttc tctggccagc
     8101 tcgatgatga tatcttcagg cacgccgtac ttttctatga tcttgttaat aaggttaatg
     8161 gcctggccga aggtcctctt caccacaggg ctcaggatga actcgtcgat catagccttc
     8221 gggatcttgt tggcggctgt caggttgatc ttcttaggct taatgttcag gtgggcgaag
     8281 atttccatct gagcccggct agaataccac tgttcttcca ggaccagccg gatgcatttc
     8341 aggctgagcc ggtgggtccc ggtgtagcca gtcagctggg cgatgttttc cttatcttcc
     8401 tcgttcagca ggatgtccag ttccgtcagc ttagatttga tgctatcctt gtcctggtag
     8461 atggtcagga tctcagcgat ctggtccagc acgtcttcgt tctccaggat gctctggtca
     8521 aacagcacgc ttttcaggtc gtggtagagc ttaaactcgg taaactgggg cttgccagac
     8581 tttgtgattc tgtagccctt gatatcttca gggttgacat tgatctcatt ggcgatctgc
     8641 ttcagtgtag gtttcttctt ctgcttgaac acgttctcga tgatgtgata cttctcatga
     8701 tactcgagct tagacagtcc atctctttgg atgaccaggt tgttcaggtc gttcagagcg
     8761 ttgaacaggt cggcactgta ggcgtatttc acgctagcca gctcatctgg gaagtaggtg
     8821 cagtggccca tcagtgtctc gtaccaagcc ttagggtcgc cttcccagcc atatggactg
     8881 cccttgccgg ggccctcgaa gtattcccgt ctcatctcaa ccagttcgat gtacttgttg
     8941 atgaagttct cgtccagctg gtggaagttc ttctgcacat tcagcagctg gatgatttcc
     9001 ttgatgatat cagcggtctt gaaccggttt ttctctcccc gcacctggcc ctcattcatt
     9061 ctttccagct ggatctggca cacaaatttg tctttcagca gcttgctgtt cttattcagc
     9121 tgttccttgg tgctgagctc gttccccacg tcgtcgttgc tgtcgatcac gtcgatcttg
     9181 tggatgcctc ttctcttggc gatatggagc agggcaatca cgagttcgtc cttgctcagg
     9241 gcttcagaca ggcccttcac gcggatggcg taggggtttg tagactgagg aatctgggac
     9301 tgatccagca gattataatc ctcgagcagc ttcttcaccc gttcaagtct gtgaatgcgg
     9361 cgtcgcttca gtcttctgct tccacgtttg gaccttcttc cctcgttgtt ttccacgttg
     9421 gcctcgggga acagtctaac gccggcatcg atgatattct ttgtctcgta gtcgatcagg
     9481 ccgtagccca cgctggtgat tccgatgtcc agtcccagta tgaatttttg gttggctgct
     9541 gggactccgt ggataccgac cttccgcttc ttctttgggg ccatttttcc cgttctagaa
     9601 aacttagatt agattgctat gctttctttc taatgagcaa gaagtaaaaa aagttgtaat
     9661 agaacaagaa aaatgaaact gaaacttgag aaattgaaga ccgtttatta acttaaatat
     9721 caatgggagg tcatcgaaag agaaaaaaat caaaaaaaaa attttcaaga aaaagaaacg
     9781 tgataaaaat ttttattgcc tttttcgacg aagaaaaaga aacgaggcgg tctctttttt
     9841 cttttccaaa cctttagtac gggtaattaa cgacacccta gaggaagaaa gaggggaaat
     9901 ttagtatgct gtgcttgggt gttttgaagt ggtacggcga tgcgcggagt ccgagaaaat
     9961 ctggaagagt aaaaaaggag tagaaacatt ttgaagctat gagcggccgc gttaacgagc
    10021 tcctttcatt tctgataaaa gtaaggcttc tc
//
