LOCUS       sequence-152577-        7605 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PmCDA1 in yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7605)
  AUTHORS   Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, 
            Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A
  TITLE     Targeted nucleotide editing using hybrid prokaryotic and vertebrate 
            adaptive immune systems.
  JOURNAL   Science. 2016 Aug 4. pii: aaf8729.
  PUBMED    27492474
REFERENCE   2  (bases 1 to 7605)
  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: "Science. 
            2016 Aug 4. pii: aaf8729."
COMMENT     Sequence Label: pRS315e_pGal-PmCDA1
FEATURES             Location/Qualifiers
     source          1..7605
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    70..573
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        610..714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(715..783,784..1575)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(933..952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1746..2334
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2488..2505
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2658..2663,2664..2681,2682..2688)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2696..2712
                     /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     2701..2723
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2720..2736
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2720..2736
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2757..2775
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2818..3482
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3034..3151
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3446..3469
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     regulatory      3483..3492
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3494..3517
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3530..4153
                     /codon_start=1
                     /product="cytidine deaminase from the sea lamprey 
                     Petromyzon marinus (Rogozin et al., 2007)"
                     /label=PmCDA1
                     /translation="MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGER
                     RACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKI
                     LEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIF
                     IQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV"
     terminator      4184..4371
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4239..4263)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      complement(4408..4453)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4489..4508)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4490..4508)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4515..4532)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4515..4531)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4524..4546)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4676..5131
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4763..4782)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4973..4994
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5431..5835
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5848..6942
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(7436..7455)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7555..7577
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata
      121 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt
      181 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa
      241 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata
      301 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct
      361 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata
      421 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact
      481 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa
      541 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg
      601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
      961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1141 ccgaaggagc taaccgcttt ttttcacaac atgggggatc atgtaactcg ccttgatcgt
     1201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1501 ggcagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1861 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     1921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2101 acgacctaca ccgaactgag atacctacag cgtgagcatt gagaaagcgc cacgcttccc
     2161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2221 agggagcttc caggggggaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc cgagcctatg gaaaaacgcc
     2341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact
     2641 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg
     2701 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcggaatt
     2761 aaccctcact aaagggaaca aaagctgggt accgggcccc ccctcgaggt cgactagttt
     2821 caaaaattct tacttttttt ttggatggac gcaaagaagt ttaataatca tattacatgg
     2881 cattaccacc atatacatat ccatatacat atccatatct aatcttactt atatgttgtg
     2941 gaaatgtaaa gagccccatt atcttagcct aaaaaaacct tctctttgga actttcagta
     3001 atacgcttaa ctgctcattg ctatattgaa gtacggatta gaagccgccg agcgggtgac
     3061 agccctccga aggaagactc tcctccgtgc gtcctcgtct tcaccggtcg cgttcctgaa
     3121 acgcagatgt gcctcgcgcc gcactgctcc gaacaataaa gattctacaa tactagcttt
     3181 tatggttatg aagaggaaaa attggcagta acctggcccc acaaaccttc aaatgaacga
     3241 atcaaattaa caaccatagg atgataatgc gattagtttt ttagccttat ttctggggta
     3301 attaatcagc gaagcgatga tttttgatct attaacagat atataaatgc aaaaactgca
     3361 taaccacttt aactaatact ttcaacattt tcggtttgta ttacttctta ttcaaatgta
     3421 ataaaagtat caacaaaaaa ttgttaatat acctctatac tttaacgtca aggagaaaaa
     3481 acccatggtg gcctggagcc acccgcagtt cgaaaaaggc ggcggatcga tgaccgacgc
     3541 tgagtacgtg agaatccatg agaagttgga catctacacg tttaagaaac agtttttcaa
     3601 caacaaaaaa tccgtgtcgc atagatgcta cgttctcttt gaattaaaac gacggggtga
     3661 acgtagagcg tgtttttggg gctatgctgt gaataaacca cagagcggga cagaacgtgg
     3721 cattcacgcc gaaatcttta gcattagaaa agtcgaagaa tacctgcgcg acaaccccgg
     3781 acaattcacg ataaattggt actcatcctg gagtccttgt gcagattgcg ctgaaaagat
     3841 cttagaatgg tataaccagg agctgcgggg gaacggccac actttgaaaa tctgggcttg
     3901 caaactctat tacgagaaaa atgcgaggaa tcaaattggg ctgtggaacc tcagagataa
     3961 cggggttggg ttgaatgtaa tggtaagtga acactaccaa tgttgcagga aaatattcat
     4021 ccaatcgtcg cacaatcaat tgaatgagaa tagatggctt gagaagactt tgaagcgagc
     4081 tgaaaaacga cggagcgagt tgtccattat gattcaggta aaaatactcc acaccactaa
     4141 gagtcctgct gtttctagag gctccggata aggatcctaa taagcgaatt tcttatgatt
     4201 tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga
     4261 ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt
     4321 gctttctcag gtatagcatg aggtcgctct tattgaccac acctctaccg gcatgccgag
     4381 caaatgcctg caaatcgctc ccgggcaaaa aaccccctca agacccgttt agaggcccca
     4441 aggggttatg ctatgcatac caagcttccg cggtggagct ccaattcgcc ctatagtgag
     4501 tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     4561 acccaactta atcgccttgc agcacatccc cccttcgcca gctggcgtaa tagcgaagag
     4621 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcgacgcg
     4681 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4741 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4801 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4861 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     4921 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4981 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     5041 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5101 aattttaaca aaatattaac gtttacaatt tcctgatgcg gtattttctc cttacgcatc
     5161 tgtgcggtat ttcacaccgc atatcgaccc tcgaggagaa cttctagtat atccacatac
     5221 ctaatattat tgccttatta aaaatggaat cggaacaatt acatcaaaat ccacattctc
     5281 ttcaaaatca attgtcctgt acttccttgt tcatgtgtgt tcaaaaacgt tatatttata
     5341 ggataattat actctatttc tcaacaagta attggttgtt tggccgagcg gtctaaggcg
     5401 cctgattcaa gaaatatctt gaccgcagtt aactgtggga atactcaggt atcgtaagat
     5461 gcaagagttc gaatctctta gcaaccatta tttttttcct caacataacg agaacacaca
     5521 ggggcgctat cgcacagaat caaattcgat gactggaaat tttttgttaa tttcagaggt
     5581 cgcctgacgc atataccttt ttcaactgaa aaattgggag aaaaaggaaa ggtgagaggc
     5641 cggaaccggc ttttcatata gaatagagaa gcgttcatga ctaaatgctt gcatcacaat
     5701 acttgaagtt gacaatatta tttaaggacc tattgttttt tccaataggt ggttagcaat
     5761 cgtcttactt tctaactttt cttacctttt acatttcagc aatatatata tatatttcaa
     5821 ggatatacca ttctaatgtc tgcccctatg tctgccccta agaagatcgt cgttttgcca
     5881 ggtgaccacg ttggtcaaga aatcacagcc gaagccatta aggttcttaa agctatttct
     5941 gatgttcgtt ccaatgtcaa gttcgatttc gaaaatcatt taattggtgg tgctgctatc
     6001 gatgctacag gtgtcccact tccagatgag gcgctggaag cctccaagaa ggttgatgcc
     6061 gttttgttag gtgctgtggg tggtcctaaa tggggtaccg gtagtgttag acctgaacaa
     6121 ggtttactaa aaatccgtaa agaacttcaa ttgtacgcca acttaagacc atgtaacttt
     6181 gcatccgact ctcttttaga cttatctcca atcaagccac aatttgctaa aggtactgac
     6241 ttcgttgttg tcagagaatt agtgggaggt atttactttg gtaagagaaa ggaagacgat
     6301 ggtgatggtg tcgcttggga tagtgaacaa tacaccgttc cagaagtgca aagaatcaca
     6361 agaatggccg ctttcatggc cctacaacat gagccaccat tgcctatttg gtccttggat
     6421 aaagctaatg ttttggcctc ttcaagatta tggagaaaaa ctgtggagga aaccatcaag
     6481 aacgaattcc ctacattgaa ggttcaacat caattgattg attctgccgc catgatccta
     6541 gttaagaacc caacccacct aaatggtatt ataatcacca gcaacatgtt tggtgatatc
     6601 atctccgatg aagcctccgt tatcccaggt tccttgggtt tgttgccatc tgcgtccttg
     6661 gcctctttgc cagacaagaa caccgcattt ggtttgtacg aaccatgcca cggttctgct
     6721 ccagatttgc caaagaataa ggttgaccct atcgccacta tcttgtctgc tgcaatgatg
     6781 ttgaaattgt cattgaactt gcctgaagaa ggtaaggcca ttgaagatgc agttaaaaag
     6841 gttttggatg caggtatcag aactggtgat ttaggtggtt ccaacagtac caccgaagtc
     6901 ggtgatgctg tcgccgaaga agttaagaaa atccttgctt aaaaagattc tcttttttta
     6961 tgatatttgt acataaactt tataaatgaa attcataata gaaacgacac gaaattacaa
     7021 aatggaatat gttcataggg tagacgaaac tatatacgca atctacatac atttatcaag
     7081 aaggagaaaa aggaggatgt aaaggaatac aggtaagcaa attgatacta atggctcaac
     7141 gtgataagga aaaagaattg cactttaaca ttaatattga caaggaggag ggcaccacac
     7201 aaaaagttag gtgtaacaga aaatcatgaa actatgattc ctaatttata tattggagga
     7261 ttttctctaa aaaaaaaaaa atacaacaaa taaaaaacac tcaatgacct gaccatttga
     7321 tggagtttaa gtcaatacct tcttgaacca tttcccataa tggtgaaagt tccctcaaga
     7381 attttactct gtcagaaacg gccttaacga cgtagtcgat atggtgcact ctcagtacaa
     7441 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     7501 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     7561 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcga
//
