LOCUS       sequence-265979-        9068 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCS4333 CEN/ARS vector, PGAL1-yeTadA1.0-100AA linker-T7 RNAP-TCYC1, 
            TRP1 selectable marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9068)
  AUTHORS   Cravens A, Jamil OK, Kong D, Sockolosky JT, Smolke CD
  TITLE     Polymerase-guided base editing enables in vivo mutagenesis and rapid
            protein engineering.
  JOURNAL   Nat Commun. 2021 Mar 11;12(1):1579. doi: 10.1038/s41467-021-21876-z.
  PUBMED    33707425
REFERENCE   2  (bases 1 to 9068)
  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: "Nat 
            Commun."; date: "2021-03-11"; volume: "12(1):1579. doi"; pages: " 
            10.1038/s41467-021-21876-z"
COMMENT     Sequence Label: pCS4333 yeTadA1.0-T7 RNAP
FEATURES             Location/Qualifiers
     source          1..9068
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(590..1381,1382..1450))
                     /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     1213..1232
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1451..1555)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    1592..2095
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     2137..2155
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2193..2215)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2315..2334
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2351..2631
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             2632..3306
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      complement(3403..3858)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3540..3561)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3752..3771
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3941..4106)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     4027..4051
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(4254..4277)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        4324..4988
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    4540..4657
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     4952..4975
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     terminator      8467..8656
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(8728..8745)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(8899..9068,1..419))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8979..8998)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
       61 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
      121 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
      181 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
      241 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
      301 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
      361 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
      421 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
      481 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
      541 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
      601 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
      661 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
      721 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
      781 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
      841 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
      901 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
      961 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     1021 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     1081 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     1141 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     1201 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     1261 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     1321 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     1381 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     1441 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     1501 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     1561 ttccccgaaa agtgccacct gggtcctttt catcacgtgc tataaaaata attataattt
     1621 aaatttttta atataaatat ataaattaaa aatagaaagt aaaaaaagaa attaaagaaa
     1681 aaatagtttt tgttttccga agatgtaaaa gactctaggg ggatcgccaa caaatactac
     1741 cttttatctt gctcttcctg ctctcaggta ttaatgccga attgtttcat cttgtctgtg
     1801 tagaagacca cacacgaaaa tcctgtgatt ttacatttta cttatcgtta atcgaatgta
     1861 tatctattta atctgctttt cttgtctaat aaatatatat gtaaagtacg ctttttgttg
     1921 aaatttttta aacctttgtt tatttttttt tcttcattcc gtaactcttc taccttcttt
     1981 atttactttc taaaatccaa atacaaaaca taaaaataaa taaacacaga gtaaattccc
     2041 aaattattcc atcattaaaa gatacgaggc gcgtgtaagt tacaggcaag cgatccgtcc
     2101 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     2161 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     2221 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     2281 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     2341 gtgcaccata aacgacatta ctatatatat aatataggaa gcatttaata gacagcatcg
     2401 taatatatgt gtactttgca gttatgacgc cagatggcag tagtggaaga tattctttat
     2461 tgaaaaatag cttgtcacct tacgtacaat cttgatccgg agcttttctt tttttgccga
     2521 ttaagaatta attcggtcga aaaaagaaaa ggagagggcc aagagggagg gcattggtga
     2581 ctattgagca cgtgagtata cgtgattaag cacacaaagg cagcttggag tatgtctgtt
     2641 attaatttca caggtagttc tggtccattg gtgaaagttt gcggcttgca gagcacagag
     2701 gccgcagaat gtgctctaga ttccgatgct gacttgctgg gtattatatg tgtgcccaat
     2761 agaaagagaa caattgaccc ggttattgca aggaaaattt caagtcttgt aaaagcatat
     2821 aaaaatagtt caggcactcc gaaatacttg gttggcgtgt ttcgtaatca acctaaggag
     2881 gatgttttgg ctctggtcaa tgattacggc attgatatcg tccaactgca tggagatgag
     2941 tcgtggcaag aataccaaga gttcctcggt ttgccagtta ttaaaagact cgtatttcca
     3001 aaagactgca acatactact cagtgcagct tcacagaaac ctcattcgtt tattcccttg
     3061 tttgattcag aagcaggtgg gacaggtgaa cttttggatt ggaactcgat ttctgactgg
     3121 gttggaaggc aagagagccc cgaaagctta cattttatgt tagctggtgg actgacgcca
     3181 gaaaatgttg gtgatgcgct tagattaaat ggcgttattg gtgttgatgt aagcggaggt
     3241 gtggagacaa atggtgtaaa agactctaac aaaatagcaa atttcgtcaa aaatgctaag
     3301 aaataggtta ttactgagta gtatttattt aagtattgtt tgtgcacttg cctgcggtgt
     3361 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata
     3421 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     3481 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     3541 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     3601 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     3661 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     3721 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     3781 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     3841 cgccgctaca gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     3901 gatcggtgcg ggcctcttcg ctattacgcc agctgaattg gagcgacctc atgctatacc
     3961 tgagaaagca acctgaccta caggaaagag ttactcaaga ataagaattt tcgttttaaa
     4021 acctaagagt cactttaaaa tttgtataca cttatttttt ttataactta tttaataata
     4081 aaaatcataa atcataagaa attcgcttat ttagaagtgt caacaacgta tctaccaacg
     4141 atttgaccct tttccatctt ttcgtaaatt tctggcaagg tagacaagcc gacaaccttg
     4201 attggagact tgaccaaacc tctggcgaag aattgttaat taagagctca gatcttatcg
     4261 tcgtcatcct tgtaatccat cgatactagt gcggccgccc tttagtgagg gttgaattcg
     4321 aattttcaaa aattcttact ttttttttgg atggacgcaa agaagtttaa taatcatatt
     4381 acatggcatt accaccatat acatatccat atacatatcc atatctaatc ttacttatat
     4441 gttgtggaaa tgtaaagagc cccattatct tagcctaaaa aaaccttctc tttggaactt
     4501 tcagtaatac gcttaactgc tcattgctat attgaagtac ggattagaag ccgccgagcg
     4561 ggtgacagcc ctccgaagga agactctcct ccgtgcgtcc tcgtcttcac cggtcgcgtt
     4621 cctgaaacgc agatgtgcct cgcgccgcac tgctccgaac aataaagatt ctacaatact
     4681 agcttttatg gttatgaaga ggaaaaattg gcagtaacct ggccccacaa accttcaaat
     4741 gaacgaatca aattaacaac cataggatga taatgcgatt agttttttag ccttatttct
     4801 ggggtaatta atcagcgaag cgatgatttt tgatctatta acagatatat aaatgcaaaa
     4861 actgcataac cactttaact aatactttca acattttcgg tttgtattac ttcttattca
     4921 aatgtaataa aagtatcaac aaaaaattgt taatatacct ctatacttta acgtcaagga
     4981 gaaaaaaccc cggatccgat gtccgaggtt gagttcagcc atgagtactg gatgagacac
     5041 gcattgactc tagcaaagag ggcgagagac gagagggaag tccccgtcgg cgccgtatta
     5101 gtcttaaata atagggtaat tggtgaaggg tggaaccgtg ctattgggtt acatgacccg
     5161 acagctcacg cggagattat ggcacttaga caaggcggtc ttgtaatgca gaactaccgt
     5221 ttaatagacg caacactgta cgtcacattc gaaccttgtg taatgtgtgc cggagccatg
     5281 atccactccc gtataggccg tgttgtcttt ggggtcagaa acgcgaagac aggagcagcc
     5341 ggttccctga tggatgtgtt acactaccct ggaatgaatc atagtgtcga gataacggag
     5401 ggtatcctgg cagacgagtg tgccgcgttg ctgtgttatt tttttaggat gccccgtcgg
     5461 gtctttaatg cgcagaaaaa agcgcagccc tctaccggct ctggaggtgg aggttctgca
     5521 gagtatgtgc gggccctctt tgactttaat gggaatgatg aagaagatct tccctttaag
     5581 aaaggagaca tcctgagaat ccgggataag cctgaagagc agtggtggaa tgcagaggac
     5641 agcgaaggaa agagggggat gattcctgtc ccttacgtgg agaagtattc cggagactat
     5701 aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga tgacaatcag
     5761 gttaacacga ttaacatagc taagaacgac ttctctgaca tcgaactggc tgctatcccg
     5821 ttcaacactc tggctgacca ttacggtgag cgtttagctc gcgaacagtt ggcccttgag
     5881 catgagtctt acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa
     5941 gctggtgagg ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag
     6001 atgattgcac gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg caagcgcccg
     6061 acagccttcc agttcctgca agaaatcaag ccggaagccg tagcgtacat caccattaag
     6121 accactctgg cttgcctaac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca
     6181 atcggtcggg ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag
     6241 cacttcaaga aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa
     6301 gcatttatgc aagttgtcga ggctgacatg ctctctaagg gtctactcgg tggcgaggcg
     6361 tggtcttcgt ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc
     6421 attgagtcaa ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac
     6481 tctgagacta tcgaactcgc acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg
     6541 ctggctggca tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc
     6601 attactggtg gtggctattg ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac
     6661 agtaagaaag cactgatgcg ctacgaagac gtttacatgc ctgaggtgta caaagcgatt
     6721 aacattgcgc aaaacaccgc atggaaaatc aacaagaaag tcctagcggt cgccaacgta
     6781 atcaccaagt ggaagcattg tccggtcgag gacatccctg cgattgagcg tgaagaactc
     6841 ccgatgaaac cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct
     6901 gccgctgctg tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag ccttgagttc
     6961 atgcttgagc aagccaataa gtttgctaac cataaggcca tctggttccc ttacaacatg
     7021 gactggcgcg gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc
     7081 aaaggactgc ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta ctactggctg
     7141 aaaatccacg gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag
     7201 ttcattgagg aaaaccacga gaacatcatg gcttgcgcta agtctccact ggagaacact
     7261 tggtgggctg agcaagattc tccgttctgc ttccttgcgt tctgctttga gtacgctggg
     7321 gtacagcacc acggcctgag ctataactgc tcccttccgc tggcgtttga cgggtcttgc
     7381 tctggcatcc agcacttctc cgcgatgctc cgagatgagg taggtggtcg cgcggttaac
     7441 ttgcttccta gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgag
     7501 attctacaag cagacgcaat caatgggacc gataacgaag tagttaccgt gaccgatgag
     7561 aacactggtg aaatctctga gaaagtcaag ctgggcacta aggcactggc tggtcaatgg
     7621 ctggcttacg gtgttactcg cagtgtgact aagcgttcag tcatgacgct ggcttacggg
     7681 tccaaagagt tcggcttccg tcaacaagtg ctggaagata ccattcagcc agctattgat
     7741 tccggcaagg gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg
     7801 atttgggaat ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa ctggcttaag
     7861 tctgctgcta agctgctggc tgctgaggtc aaagataaga agactggaga gattcttcgc
     7921 aagcgttgcg ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca ggaatacaag
     7981 aagcctattc agacgcgctt gaacctgatg ttcctcggtc agttccgctt acagcctacc
     8041 attaacacca acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct
     8101 aactttgtac acagccaaga cggtagccac cttcgtaaga ctgtagtgtg ggcacacgag
     8161 aagtacggaa tcgaatcttt tgcactgatt cacgactcct tcggtaccat tccggctgac
     8221 gctgcgaacc tgttcaaagc agtgcgcgaa actatggttg acacatatga gtcttgtgat
     8281 gtactggctg atttctacga ccagttcgct gaccagttgc acgagtctca attggacaaa
     8341 atgccagcac ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc
     8401 gcgttcgcgt aaaatcagta agcttggtac cgcggctagc taagcttggt accgcggcta
     8461 gctaagatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat
     8521 ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt
     8581 ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt
     8641 tttgggacgc tcgaagatcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     8701 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     8761 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     8821 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     8881 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     8941 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     9001 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     9061 ctttctcc
//
