LOCUS       sequence-439310-        5544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Acceptor plasmid for yeast retron-Eco1 ncRNA variant libraries..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5544)
  AUTHORS   Crawford KD, Khan AG, Lopez SC, Goodarzi H, Shipman SL
  TITLE     High throughput variant libraries and machine learning yield design 
            rules for retron gene editors.
  JOURNAL   Nucleic Acids Res. 2025 Jan 11;53(2):gkae1199. doi: 
            10.1093/nar/gkae1199.
  PUBMED    39658047
REFERENCE   2  (bases 1 to 5544)
  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: "Nucleic 
            Acids Res."; date: "2025-01-11"; volume: "53(2):gkae1199. doi"; 
            pages: " 10.1093/nar/gkae1199"
COMMENT     Sequence Label: pKDC.100
FEATURES             Location/Qualifiers
     source          1..5544
                     /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"
     primer_bind     complement(3078..3097)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3079..3097)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3107..3124)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3107..3123)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3116..3138)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3264..3719
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3351..3370)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3561..3582
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3850..4653)
                     /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(4654..4869)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4893..4912)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5012..5034
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5072..5090)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(5131..5544,1..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 agttttcatt 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 tttgctcaag
     1921 tagagggggt aatttttccc ctttattttg ttcatacatt cttaaattgc tttgcctctc
     1981 cttttggaaa gctatacttc ggagcactgt tgagcgaagg ctcattagat atattttctg
     2041 tcattttcct taacccaaaa ataagggaaa gggtccaaaa agcgctcgga caactgttga
     2101 ccgtgatccg aaggactggc tatacagtgt tcacaaaata gccaagctga aaataatgtg
     2161 tagctatgtt cagttagttt ggctagcaaa gatataaaag caggtcggaa atatttatgg
     2221 gcattattat gcagagcatc aacatgataa aaaaaaacag ttgaatattc cctcaaaagg
     2281 gtgcgcatct gatgagtccg tgaggacgaa acgagctagc tcgtcatgat aagattccgt
     2341 atgcgcaccc ttagcgagag gtttatcatt aaggtcaacc tctggatgtt gtttcggcat
     2401 cctgcattga atctgagtta ctgtctgttt tccttgatgc aggtgtagct tgatgcggaa
     2461 ggagatctgg aatgaactgg tctatgtgac agaaactgtg caagtaccta atctcgttag
     2521 tgtaggttct agctacacct gcatcagttt cagagctatg ctggaaacag catagcaagt
     2581 tgaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttgat
     2641 ggccggcatg gtcccagcct cctcgctggc gccggctggg caacaccttc gggtggcgaa
     2701 tgggacttaa agaaagtgga atattcattc atatcatatt ttttctatta actgcctggt
     2761 ttcttttaaa ttttttattg gttgtcgact tgaacggagt gacaatatat atatatatat
     2821 ttaataatga catcattatc tgtaaatctg attcttaatg ctattctagt tatgtaagag
     2881 tggtcctttc cataaaaaaa aaaaaaaaga aaaaagaatt ttaggaatac aatgcagctt
     2941 gtaagtaaaa tctggaatat tcatatcgcc acaacttctt atgcttataa aagcactaat
     3001 gcctgaattt atgttgaaaa tatgtgtcac aaataaagaa actgtgacat ctgacacatt
     3061 tccacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     3121 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     3181 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     3241 tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3301 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3361 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3421 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3481 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3541 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3601 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3661 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc
     3721 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat agggtaataa
     3781 ctgatataat taaattgaag ctctaatttg tgagtttagt atacatgcat ttacttataa
     3841 tacagttttt tagttttgct ggccgcatct tctcaaatat gcttcccagc ctgcttttct
     3901 gtaacgttca ccctctacct tagcatccct tccctttgca aatagtcctc ttccaacaat
     3961 aataatgtca gatcctgtag agaccacatc atccacggtt ctatactgtt gacccaatgc
     4021 gtctcccttg tcatctaaac ccacaccggg tgtcataatc aaccaatcgt aaccttcatc
     4081 tcttccaccc atgtctcttt gagcaataaa gccgataaca aaatctttgt cgctcttcgc
     4141 aatgtcaaca gtacccttag tatattctcc agtagatagg gagcccttgc atgacaattc
     4201 tgctaacatc aaaaggcctc taggttcctt tgttacttct tctgccgcct gcttcaaacc
     4261 gctaacaata cctgggccca ccacaccgtg tgcattcgta atgtctgccc attctgctat
     4321 tctgtataca cccgcagagt actgcaattt gactgtatta ccaatgtcag caaattttct
     4381 gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc tttagcggct taactgtgcc
     4441 ctccatggaa aaatcagtca agatatccac atgtgttttt agtaaacaaa ttttgggacc
     4501 taatgcttca actaactcca gtaattcctt ggtggtacga acatccaatg aagcacacaa
     4561 gtttgtttgc ttttcgtgca tgatattaaa tagcttggca gcaacaggac taggatgagt
     4621 agcagcacgt tccttatatg tagctttcga catgatttat cttcgtttcc tgcaggtttt
     4681 tgttctgtgc agttgggtta agaatactgg gcaatttcat gtttcttcaa cactacatat
     4741 gcgtatatat accaatctaa gtctgtgctc cttccttcgt tcttccttct gttcggagat
     4801 taccgaatca aaaaaatttc aaggaaaccg aaatcaaaaa aaagaataaa aaaaaaatga
     4861 tgaattgaaa aggtggtatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     4921 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4981 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5041 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5101 aatgtcatga taataatggt ttcttagacg gatcgcttgc ctgtaactta cacgcgcctc
     5161 gtatctttta atgatggaat aatttgggaa tttactctgt gtttatttat ttttatgttt
     5221 tgtatttgga ttttagaaag taaataaaga aggtagaaga gttacggaat gaagaaaaaa
     5281 aaataaacaa aggtttaaaa aatttcaaca aaaagcgtac tttacatata tatttattag
     5341 acaagaaaag cagattaaat agatatacat tcgattaacg ataagtaaaa tgtaaaatca
     5401 caggattttc gtgtgtggtc ttctacacag acaagatgaa acaattcggc attaatacct
     5461 gagagcagga agagcaagat aaaaggtagt atttgttggc gatcccccta gagtctttta
     5521 catcttcgga aaacaaaaac tatt
//
