LOCUS       BSR-sg_BHM2680          6743 bp ds-DNA     circular     30-MAY-2025
DEFINITION  .
KEYWORDS    "marker:HygR, AmpR"
COMMENT     pRS316-PCnU6-scaffold, contains template for amplification of PCnU6
            and scaffold for fusion PCR. <small>[1] Short-homology genome
            engineering in Cryptococcus neoformans Unpublished</small>
FEATURES             Location/Qualifiers
     CDS             complement(171..962)
                     /label="AmpR"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="codon_start: 1 gene: bla note: confers resistance to ampicillin, carbenicillin, and related antibiotics product: β-lactamase transl_table: 1 translation: MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW*"
     CDS             complement(963..1031)
                     /label="AmpR"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="codon_start: 1 gene: bla note: confers resistance to ampicillin, carbenicillin, and related antibiotics product: β-lactamase transl_table: 1 translation: MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW*"
     promoter        complement(1032..1136)
                     /label="AmpR promoter"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="gene: bla"
     misc_feature    1173..1676
                     /label="CEN/ARS"
                     /ApEinfo_revcolor="#454f59"
                     /ApEinfo_fwdcolor="#454f59"
                     /note="note: S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence"
     promoter        1938..2153
                     /label="URA3 promoter"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="gene: S. cerevisiae URA3"
     CDS             2154..2957
                     /label="URA3"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
                     /note="codon_start: 1 gene: S. cerevisiae URA3 note: yeast auxotrophic marker, counterselectable with 5-fluoroorotic acid (5-FOA) product: orotidine-5'-phosphate decarboxylase, required for uracil biosynthesis transl_table: 1 translation: MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELLELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQYSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGEYTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDDVVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN*"
     rep_origin      complement(3088..3543)
                     /label="f1 ori"
                     /ApEinfo_revcolor="#ffff00"
                     /ApEinfo_fwdcolor="#ffff00"
                     /note="direction: LEFT note: f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis"
     primer_bind     3684..3700
                     /label="M13 fwd"
                     /ApEinfo_revcolor="#a020f0"
                     /ApEinfo_fwdcolor="#a020f0"
                     /note="note: common sequencing primer, one of multiple similar variants"
     promoter        3707..3725
                     /label="T7 promoter"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="note: promoter for bacteriophage T7 RNA polymerase"
     misc_feature    3740..3757
                     /label="Adaptor for KO F"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     misc_feature    complement(3744..3762)
                     /label="MJB555"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     misc_feature    3770..4512
                     /label="ACT1 Promoter"
                     /ApEinfo_revcolor="#c6c9d1"
                     /ApEinfo_fwdcolor="#c6c9d1"
     misc_feature    4531..5005
                     /label="BSR_CnOpt_Intron"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
     misc_feature    4672..4723
                     /label="Intron"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     misc_feature    5033..5259
                     /label="TRP1 Terminator"
                     /ApEinfo_revcolor="#b4abac"
                     /ApEinfo_fwdcolor="#b4abac"
     misc_feature    5167..5185
                     /label="MJB556"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     misc_feature    complement(5196..5217)
                     /label="Adaptor for KO R"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
     promoter        5260..5547
                     /label="CnU6 promoter"
                     /ApEinfo_revcolor="#f8d3a9"
                     /ApEinfo_fwdcolor="#f8d3a9"
     misc_RNA        5576..5651
                     /label="gRNA scaffold"
                     /ApEinfo_revcolor="#00ccff"
                     /ApEinfo_fwdcolor="#00ccff"
                     /note="note: guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system"
     primer_bind     complement(5669..5685)
                     /label="KS primer"
                     /ApEinfo_revcolor="#a020f0"
                     /ApEinfo_fwdcolor="#a020f0"
                     /note="note: common sequencing primer, one of multiple similar variants"
     promoter        complement(5714..5732)
                     /label="T3 promoter"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="note: promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5753..5769)
                     /label="M13 rev"
                     /ApEinfo_revcolor="#a020f0"
                     /ApEinfo_fwdcolor="#a020f0"
                     /note="note: common sequencing primer, one of multiple similar variants"
     protein_bind    5777..5793
                     /label="lac operator"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="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-β-D-thiogalactopyranoside (IPTG)."
     promoter        complement(5801..5807)
                     /label="lac promoter"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="note: promoter for the E. coli lac operon"
     promoter        complement(5808..5825)
                     /label="lac promoter"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="note: promoter for the E. coli lac operon"
     promoter        complement(5826..5831)
                     /label="lac promoter"
                     /ApEinfo_revcolor="#b7e6d7"
                     /ApEinfo_fwdcolor="#b7e6d7"
                     /note="note: promoter for the E. coli lac operon"
     protein_bind    5846..5867
                     /label="CAP binding site"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="bound_moiety: E. coli catabolite activator protein note: CAP binding activates transcription in the presence of cAMP."
     rep_origin      complement(6155..6743)
                     /label="ori"
                     /ApEinfo_revcolor="#ffff00"
                     /ApEinfo_fwdcolor="#ffff00"
                     /note="direction: LEFT note: high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgggtccttt tcatcacgtg ctataaaaat aattataatt
     1201 taaatttttt aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa
     1261 aaaatagttt ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta
     1321 ccttttatct tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt
     1381 gtagaagacc acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt
     1441 atatctattt aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt
     1501 gaaatttttt aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt
     1561 tatttacttt ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc
     1621 caaattattc catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatccgtc
     1681 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     1741 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     1801 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     1861 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     1921 gtgcaccata ccaccttttc aattcatcat ttttttttta ttcttttttt tgatttcggt
     1981 ttccttgaaa tttttttgat tcggtaatct ccgaacagaa ggaagaacga aggaaggagc
     2041 acagacttag attggtatat atacgcatat gtagtgttga agaaacatga aattgcccag
     2101 tattcttaac ccaactgcac agaacaaaaa cctgcaggaa acgaagataa atcatgtcga
     2161 aagctacata taaggaacgt gctgctactc atcctagtcc tgttgctgcc aagctattta
     2221 atatcatgca cgaaaagcaa acaaacttgt gtgcttcatt ggatgttcgt accaccaagg
     2281 aattactgga gttagttgaa gcattaggtc ccaaaatttg tttactaaaa acacatgtgg
     2341 atatcttgac tgatttttcc atggagggca cagttaagcc gctaaaggca ttatccgcca
     2401 agtacaattt tttactcttc gaagacagaa aatttgctga cattggtaat acagtcaaat
     2461 tgcagtactc tgcgggtgta tacagaatag cagaatgggc agacattacg aatgcacacg
     2521 gtgtggtggg cccaggtatt gttagcggtt tgaagcaggc ggcagaagaa gtaacaaagg
     2581 aacctagagg ccttttgatg ttagcagaat tgtcatgcaa gggctcccta tctactggag
     2641 aatatactaa gggtactgtt gacattgcga agagcgacaa agattttgtt atcggcttta
     2701 ttgctcaaag agacatgggt ggaagagatg aaggttacga ttggttgatt atgacacccg
     2761 gtgtgggttt agatgacaag ggagacgcat tgggtcaaca gtatagaacc gtggatgatg
     2821 tggtctctac aggatctgac attattattg ttggaagagg actatttgca aagggaaggg
     2881 atgctaaggt agagggtgaa cgttacagaa aagcaggctg ggaagcatat ttgagaagat
     2941 gcggccagca aaactaaaaa actgtattat aagtaaatgc atgtatacta aactcacaaa
     3001 ttagagcttc aatttaatta tatcagttat taccctatgc ggtgtgaaat accgcacaga
     3061 tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt taatattttg ttaaaattcg
     3121 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     3181 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     3241 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     3301 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     3361 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     3421 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     3481 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     3541 cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     3601 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     3661 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattGTAAT ACGACTCACT
     3721 ATAGGGCGAA TTGGagctcg catgcaggat tcgagtggca tggtgtgcac tgagtgtatg
     3781 gttgtcggag gagaggatga tggtaacaac aatagcagca acgtcactcg acgcgcgtcc
     3841 ggtgtgccac acggggtaac gccgagtcgc cgtcagggtc gccgagacca ctctcacagc
     3901 gtcaccgttg gcaccagctc agcttacagc ttctatcctc cgccagcatc cacatacatc
     3961 ccctataccg catcccccac ccactgccca aggtgagtca tcttcccgcc cccttccctt
     4021 gcccgccact cagtcctcca tcctccacta atccacctta tcgcacccac cgcctatcgc
     4081 acatccgagc acaatgctgg gcctgccagg ggctgctaga tggtgctctc cccacgctga
     4141 tctgcatgcc ggccattgga tcatgggtgc taggtgctgg gtgctggatg ttggatgctg
     4201 gatgctgggt gcacgcttgg tcatttcctt ccaggattga cggtcgccga gaggacgacg
     4261 tggcgttcga caacgagggc cgatagcacc gcatcgcctc gacctgcatc catctcgcct
     4321 tgtccttttg gtgcaacaat ccatccgtgc tggtgccaca cgcatagctg gaagagatgg
     4381 atgtgcgttg aacagagctg ccgtcaggac tttttggtgc acggacccta ttgtcctccc
     4441 caatcttcac cgcgtctcct aatatgcagc ctctttgcta attgtctttt tccattagta
     4501 aactcgccca acatgtctat ggcggccgcc atgaagacct tcaacatctc ccagcaggac
     4561 ctcgagctcg tcgaggtcgc caccgagaag atcaccatgc tctacgagga caacaagcac
     4621 cacgtcggtg ccgccatccg aaccaagacc ggtgagatca tctccgccgt cgtatgtaac
     4681 cccgtttccc gcaacatgtt actccactaa catctcccga cagcacatcg aggcctacat
     4741 cggtcgagtc accgtctgtg ccgaggccat cgccatcggt tccgccgtct ccaacggtca
     4801 gaaggacttc gacaccatcg tcgccgtccg acacccctac tccgacgagg tcgaccgatc
     4861 catccgagtc gtctccccct gtggtatgtg tcgagagctc atctccgact acgcccccga
     4921 ctgtttcgtc ctcatcgaga tgaacggtaa gctcgtcaag accaccatcg aggagctcat
     4981 ccccctcaag tacacccgaa actaagaatt cgtgaaggcg gtaaggggtt aattttcctt
     5041 agagggtgta tatatacata ttagagaagt gatacaattt tagcacactg cgaattcgag
     5101 acagacatcg tgtcaatcat cttttttgac attatatgcc cattaatcta tctacagaca
     5161 acaataccat ccttcccacc ctcagcaacg ccgttgaatc ctcaggatct tcatggctcc
     5221 ttgtctctga aaccaggaag ctagtttcta catctcttcA CTAGTGGATC CCCCTTGCAT
     5281 TAGAACTAAA AACAAAGCAt gattattaca gttcatttat tttttaaatt gatcggcatg
     5341 catgcaaagt atacgtgcaa ggacaatggt aacctgcagg tgtgaccgat aattataacc
     5401 atttgttgag aatgaagagg tgaggagaaa aacaatggat gacgggaaaa aaataaaaaa
     5461 acactgagac ggcgtggacc gccgtcttat ttgcttccgt tatccgccaa agtggaaatt
     5521 gcacatacac cggcagggta tactgttGCT AGATGCGAGA TATCCTCATG CCTAggtttt
     5581 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     5641 cgagtcggtg cttttttatc aagcttatcg ataccgtcga cctcgagggg gggccGggta
     5701 ccagcttttg ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc
     5761 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5821 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5881 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5941 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     6001 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6061 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6121 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6181 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6241 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6301 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6361 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6421 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6481 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6541 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6601 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6661 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6721 cgcgcagaaa aaaaggatct caa
//