LOCUS       sequence-435230-        6582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Ade2, Can1 and Faa1 retron msd donor-gRNAs from a single 
            Gal7 promoter, separated by a Csy4 recognition sequence, with an msr
            expressed in trans.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6582)
  AUTHORS   Gonzalez-Delgado A, Lopez SC, Rojas-Montero M, Fishman CB, Shipman 
            SL
  TITLE     Simultaneous multi-site editing of individual genomes using retron 
            arrays.
  JOURNAL   Nat Chem Biol. 2024 Nov;20(11):1482-1492. doi: 
            10.1038/s41589-024-01665-7. Epub 2024 Jul 9.
  PUBMED    38982310
REFERENCE   2  (bases 1 to 6582)
  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"; doi: 
            "10.1038/s41589-024-01665-7"; journalName: "Nat Chem Biol"; date: 
            "2024-11"; volume: "20"; issue: "11"; pages: "1482-1492"
COMMENT     Sequence Label: pSCL475
FEATURES             Location/Qualifiers
     source          1..6582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        127..231
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(232..300,301..1092)
                     /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     complement(450..469)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1263..1851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1752..1771
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2290..2309
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    2599..2618
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    2912..2931
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    3225..3244
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     complement(3622..3641)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3623..3641)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3651..3668)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3651..3667)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3660..3682)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3738..3754
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3738..3754
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3773..3793
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3775..3793
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3811..4079
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     terminator      4177..4196
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      4302..4757
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4389..4408)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4599..4620
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4888..5691)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(5692..5907)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5931..5950)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6050..6072
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6110..6128)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(6169..6582,1..90)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 ttttctttaa tttctttttt tactttctat ttttaattta tatatttata ttaaaaaatt
       61 taaattataa ttatttttat agcacgtgat gaaaaggacc caggtggcac ttttcgggga
      121 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
      181 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
      241 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
      301 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
      361 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
      421 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
      481 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
      541 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
      601 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
      661 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
      721 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
      781 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
      841 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
      901 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
      961 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     1021 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     1081 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     1141 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     1201 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     1261 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     1321 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     1381 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     1441 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     1501 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     1561 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     1621 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     1681 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     1741 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     1801 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaaggccagc
     1861 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctggcct tttgctggcc
     1921 ttttgctcaa gtagaggggg taatttttcc cctttatttt gttcatacat tcttaaattg
     1981 ctttgcctct ccttttggaa agctatactt cggagcactg ttgagcgaag gctcattaga
     2041 tatattttct gtcattttcc ttaacccaaa aataagggaa agggtccaaa aagcgctcgg
     2101 acaactgttg accgtgatcc gaaggactgg ctatacagtg ttcacaaaat agccaagctg
     2161 aaaataatgt gtagctatgt tcagttagtt tggctagcaa agatataaaa gcaggtcgga
     2221 aatatttatg ggcattatta tgcagagcat caacatgata aaaaaaaaca gttgaatatt
     2281 ccctcaaaag ttcactgccg tataggcagc taagaaatct gagttactgt ctgttttcct
     2341 tggaaatgtt ctatttagaa acaggggaat tgcttattaa cgaaattgcc tgaaggcctc
     2401 acaactctgg acattatacc attgatgctt gcgtcacttc aggaaacccg tttcttctga
     2461 cgtaagggtg cgcatacgga atcttatcaa ttaacgaaat tgccccagtt tcagagctat
     2521 gctggaaaca gcatagcaag ttgaaataag gctagtccgt tatcaacttg aaaaagtggc
     2581 accgagtcgg tgctttttgt tcactgccgt ataggcagct aagaaaccta tctgagttac
     2641 tgtctgtttt cctgcagttt tcgttactgc tgcatttggc gctttggctt acatggagac
     2701 atctacttga ggtgacaaag ttttcgaatg gctattaaat atcactggtg ttgaggaaac
     2761 ccgtttcttc tgacgtaagg gtgcgcatac ggaatcttat cattacatgg agacatctac
     2821 gtttcagagc tatgctggaa acagcatagc aagttgaaat aaggctagtc cgttatcaac
     2881 ttgaaaaagt ggcaccgagt cggtgctttt tgttcactgc cgtataggca gctaagaaac
     2941 ctatctgagt tactgtctgt tttcctcagg attctttacc tagcttcaag atgtcgtcaa
     3001 agctaaaggt cttgatatct catctaactt ctttaattct gttgatggca tcatgagcag
     3061 attgatagga aacccgtttc ttctgacgta agggtgcgca tacggaatct tatcagctaa
     3121 aggtcttgat atcgtttcag agctatgctg gaaacagcat agcaagttga aataaggcta
     3181 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttgttcac tgccgtatag
     3241 gcagctaaga aaaaagaaag tggaatattc attcatatca tattttttct attaactgcc
     3301 tggtttcttt taaatttttt attggttgtc gacttgaacg gagtgacaat atatatatat
     3361 atatttaata atgacatcat tatctgtaaa tctgattctt aatgctattc tagttatgta
     3421 agagtggtcc tttccataaa aaaaaaaaaa aagaaaaaag aattttagga atacaatgca
     3481 gcttgtaagt aaaatctgga atattcatat cgccacaact tcttatgctt ataaaagcac
     3541 taatgcctga atttatgttg aaaatatgtg tcacaaataa agaaactgtg acatctgaca
     3601 catttccacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     3661 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     3721 catccccctt tcgccagcag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     3781 ccctcactaa agggaacaaa agctggagct tctttgaaaa gataatgtat gattatgctt
     3841 tcactcatat ttatacagaa acttgatgtt ttctttcgag tatatacaag gtgattacat
     3901 gtacgtttga agtacaactc tagattttgt agtgccctct tgggctagcg gtaaaggtgc
     3961 gcattttttc acaccctaca atgttctgtt caaaagattt tggtcaaacg ctgtagaagt
     4021 gaaagttggt gcgcatgttt cggcgttcga aacttctccg cagtgaaaga taaatgatct
     4081 gataagattc cgtaatgcgc acccttagcg agaggtttat cattaaggtc aacctctgga
     4141 tgttgtttcg gcatcctgca ttgaatctga gttacttttt tttgtttttt atgtcttcga
     4201 gtcatgtaat tagttatgtc acgcttacgt tctggcgtaa tagcgaagag gcccgcaccg
     4261 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gacgcgccct gtagcggcgc
     4321 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4381 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4441 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4501 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4561 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4621 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     4681 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4741 attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4801 caccgcatag ggtaataact gatataatta aattgaagct ctaatttgtg agtttagtat
     4861 acatgcattt acttataata cagtttttta gttttgctgg ccgcatcttc tcaaatatgc
     4921 ttcccagcct gcttttctgt aacgttcacc ctctacctta gcatcccttc cctttgcaaa
     4981 tagtcctctt ccaacaataa taatgtcaga tcctgtagag accacatcat ccacggttct
     5041 atactgttga cccaatgcgt ctcccttgtc atctaaaccc acaccgggtg tcataatcaa
     5101 ccaatcgtaa ccttcatctc ttccacccat gtctctttga gcaataaagc cgataacaaa
     5161 atctttgtcg ctcttcgcaa tgtcaacagt acccttagta tattctccag tagataggga
     5221 gcccttgcat gacaattctg ctaacatcaa aaggcctcta ggttcctttg ttacttcttc
     5281 tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc acaccgtgtg cattcgtaat
     5341 gtctgcccat tctgctattc tgtatacacc cgcagagtac tgcaatttga ctgtattacc
     5401 aatgtcagca aattttctgt cttcgaagag taaaaaattg tacttggcgg ataatgcctt
     5461 tagcggctta actgtgccct ccatggaaaa atcagtcaag atatccacat gtgtttttag
     5521 taaacaaatt ttgggaccta atgcttcaac taactccagt aattccttgg tggtacgaac
     5581 atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg atattaaata gcttggcagc
     5641 aacaggacta ggatgagtag cagcacgttc cttatatgta gctttcgaca tgatttatct
     5701 tcgtttcctg caggtttttg ttctgtgcag ttgggttaag aatactgggc aatttcatgt
     5761 ttcttcaaca ctacatatgc gtatatatac caatctaagt ctgtgctcct tccttcgttc
     5821 ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa ggaaaccgaa atcaaaaaaa
     5881 agaataaaaa aaaaatgatg aattgaaaag gtggtatggt gcactctcag tacaatctgc
     5941 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     6001 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     6061 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     6121 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgga tcgcttgcct
     6181 gtaacttaca cgcgcctcgt atcttttaat gatggaataa tttgggaatt tactctgtgt
     6241 ttatttattt ttatgttttg tatttggatt ttagaaagta aataaagaag gtagaagagt
     6301 tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa tttcaacaaa aagcgtactt
     6361 tacatatata tttattagac aagaaaagca gattaaatag atatacattc gattaacgat
     6421 aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt ctacacagac aagatgaaac
     6481 aattcggcat taatacctga gagcaggaag agcaagataa aaggtagtat ttgttggcga
     6541 tccccctaga gtcttttaca tcttcggaaa acaaaaacta tt
//
