LOCUS       sequence-435229-        5664 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector for library cloning of msd-sgRNA arrays: 
            GAL7p-Csy4-PaqCI-PaqCI-GAL7t // SNR52p-msr-SUP4t.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5664)
  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 5664)
  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: pSCL452
FEATURES             Location/Qualifiers
     source          1..5664
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(384..403)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(385..403)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(413..430)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(413..429)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(422..444)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     500..516
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     500..516
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     535..555
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        537..555
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        573..841
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     terminator      939..958
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      1064..1519
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1151..1170)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1361..1382
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1650..2453)
                     /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(2454..2669)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(2693..2712)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2812..2834
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2872..2890)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    2931..3434
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        3471..3575
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3576..3644,3645..4436)
                     /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(3794..3813)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4607..5195
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5096..5115
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5623..5642
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
ORIGIN
        1 tatcacctgc actaaaagaa agtggaatat tcattcatat catatttttt ctattaactg
       61 cctggtttct tttaaatttt ttattggttg tcgacttgaa cggagtgaca atatatatat
      121 atatatttaa taatgacatc attatctgta aatctgattc ttaatgctat tctagttatg
      181 taagagtggt cctttccata aaaaaaaaaa aaaagaaaaa agaattttag gaatacaatg
      241 cagcttgtaa gtaaaatctg gaatattcat atcgccacaa cttcttatgc ttataaaagc
      301 actaatgcct gaatttatgt tgaaaatatg tgtcacaaat aaagaaactg tgacatctga
      361 cacatttcca cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg
      421 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
      481 cacatccccc tttcgccagc aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
      541 aaccctcact aaagggaaca aaagctggag cttctttgaa aagataatgt atgattatgc
      601 tttcactcat atttatacag aaacttgatg ttttctttcg agtatataca aggtgattac
      661 atgtacgttt gaagtacaac tctagatttt gtagtgccct cttgggctag cggtaaaggt
      721 gcgcattttt tcacacccta caatgttctg ttcaaaagat tttggtcaaa cgctgtagaa
      781 gtgaaagttg gtgcgcatgt ttcggcgttc gaaacttctc cgcagtgaaa gataaatgat
      841 ctgataagat tccgtaatgc gcacccttag cgagaggttt atcattaagg tcaacctctg
      901 gatgttgttt cggcatcctg cattgaatct gagttacttt tttttgtttt ttatgtcttc
      961 gagtcatgta attagttatg tcacgcttac gttctggcgt aatagcgaag aggcccgcac
     1021 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc
     1081 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1141 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1201 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1261 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1321 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1381 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     1441 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     1501 atattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     1561 cacaccgcat agggtaataa ctgatataat taaattgaag ctctaatttg tgagtttagt
     1621 atacatgcat ttacttataa tacagttttt tagttttgct ggccgcatct tctcaaatat
     1681 gcttcccagc ctgcttttct gtaacgttca ccctctacct tagcatccct tccctttgca
     1741 aatagtcctc ttccaacaat aataatgtca gatcctgtag agaccacatc atccacggtt
     1801 ctatactgtt gacccaatgc gtctcccttg tcatctaaac ccacaccggg tgtcataatc
     1861 aaccaatcgt aaccttcatc tcttccaccc atgtctcttt gagcaataaa gccgataaca
     1921 aaatctttgt cgctcttcgc aatgtcaaca gtacccttag tatattctcc agtagatagg
     1981 gagcccttgc atgacaattc tgctaacatc aaaaggcctc taggttcctt tgttacttct
     2041 tctgccgcct gcttcaaacc gctaacaata cctgggccca ccacaccgtg tgcattcgta
     2101 atgtctgccc attctgctat tctgtataca cccgcagagt actgcaattt gactgtatta
     2161 ccaatgtcag caaattttct gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc
     2221 tttagcggct taactgtgcc ctccatggaa aaatcagtca agatatccac atgtgttttt
     2281 agtaaacaaa ttttgggacc taatgcttca actaactcca gtaattcctt ggtggtacga
     2341 acatccaatg aagcacacaa gtttgtttgc ttttcgtgca tgatattaaa tagcttggca
     2401 gcaacaggac taggatgagt agcagcacgt tccttatatg tagctttcga catgatttat
     2461 cttcgtttcc tgcaggtttt tgttctgtgc agttgggtta agaatactgg gcaatttcat
     2521 gtttcttcaa cactacatat gcgtatatat accaatctaa gtctgtgctc cttccttcgt
     2581 tcttccttct gttcggagat taccgaatca aaaaaatttc aaggaaaccg aaatcaaaaa
     2641 aaagaataaa aaaaaaatga tgaattgaaa aggtggtatg gtgcactctc agtacaatct
     2701 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     2761 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     2821 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     2881 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg gatcgcttgc
     2941 ctgtaactta cacgcgcctc gtatctttta atgatggaat aatttgggaa tttactctgt
     3001 gtttatttat ttttatgttt tgtatttgga ttttagaaag taaataaaga aggtagaaga
     3061 gttacggaat gaagaaaaaa aaataaacaa aggtttaaaa aatttcaaca aaaagcgtac
     3121 tttacatata tatttattag acaagaaaag cagattaaat agatatacat tcgattaacg
     3181 ataagtaaaa tgtaaaatca caggattttc gtgtgtggtc ttctacacag acaagatgaa
     3241 acaattcggc attaatacct gagagcagga agagcaagat aaaaggtagt atttgttggc
     3301 gatcccccta gagtctttta catcttcgga aaacaaaaac tattttttct ttaatttctt
     3361 tttttacttt ctatttttaa tttatatatt tatattaaaa aatttaaatt ataattattt
     3421 ttatagcacg tgatgaaaag gacccaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3481 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3541 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3601 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3661 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3721 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3781 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     3841 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3901 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3961 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     4021 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     4081 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     4141 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     4201 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4261 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4321 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4381 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4441 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4501 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4561 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4621 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4681 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4741 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4801 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4861 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4921 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4981 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5041 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5101 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5161 tgtgatgctc gtcagggggg cggagcctat ggaaaaaggc cagcaacgcg gcctttttac
     5221 ggttcctggc cttttgctgg ccttttgctg gccttttgct caagtagagg gggtaatttt
     5281 tcccctttat tttgttcata cattcttaaa ttgctttgcc tctccttttg gaaagctata
     5341 cttcggagca ctgttgagcg aaggctcatt agatatattt tctgtcattt tccttaaccc
     5401 aaaaataagg gaaagggtcc aaaaagcgct cggacaactg ttgaccgtga tccgaaggac
     5461 tggctataca gtgttcacaa aatagccaag ctgaaaataa tgtgtagcta tgttcagtta
     5521 gtttggctag caaagatata aaagcaggtc ggaaatattt atgggcatta ttatgcagag
     5581 catcaacatg ataaaaaaaa acagttgaat attccctcaa aagttcactg ccgtataggc
     5641 agctaagaaa cagggcaggt gctg
//
