LOCUS       sequence-376702-        7086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Test effect of extending -Eco2 a1/a2 in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7086)
  AUTHORS   Lopez SC, Crawford KD, Lear SK, Bhattarai-Kline S, Shipman SL
  TITLE     Precise genome editing across kingdoms of life using retron-derived 
            DNA.
  JOURNAL   Nat Chem Biol. 2022 Feb;18(2):199-206. doi: 
            10.1038/s41589-021-00927-y. Epub 2021 Dec 23.
  PUBMED    34949838
REFERENCE   2  (bases 1 to 7086)
  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/s41589-021-00927-y"; journalName: "Nat Chem Biol"; date: 
            "2022-02"; volume: "18"; issue: "2"; pages: "199-206"
COMMENT     Sequence Label: pSCL.031
FEATURES             Location/Qualifiers
     source          1..7086
                     /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(4620..4639)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4621..4639)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4649..4666)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4649..4665)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4658..4680)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4806..5261
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4893..4912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5103..5124
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5392..6195)
                     /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(6196..6411)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6435..6454)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6554..6576
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6614..6632)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(6673..7086,1..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 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 cctcaaaacg
     2281 ccacatgaca aaaacttcaa agctggatgc gctgcgggcg gctactagta gggaagattt
     2341 ggcgaagatt ctcgacataa agttggtgtt tctgacaaac gtgttgtacc gcataggatc
     2401 cgacaaccag tatacgcaat tcacaatacc caaaaagggt aaaggtgtcc gcaccatcag
     2461 cgcaccaacg gaccgactta aggatataca gaggaggatt tgtgatcttc ttagtgactg
     2521 tagggatgaa atctttgcga ttaggaagat ctctaataat tactcattcg gcttcgaaag
     2581 aggaaaatca attatactca atgcttacaa gcatcgaggg aagcaaatta tattgaacat
     2641 cgaccttaag gacttctttg agagctttaa ctttgggaga gtccgggggt actttctctc
     2701 caaccaggac ttcttgttga acccagttgt ggcaacaacg ttggcgaagg ccgcctgcta
     2761 caacgggact ctgcctcagg ggtccccatg ttcccctatt ataagtaacc ttatctgtaa
     2821 cattatggac atgcggctcg caaagctcgc caagaagtac ggctgcactt atagtcgata
     2881 tgcggatgac attacgatca gcaccaataa aaataccttc ccgttggaga tggcgactgt
     2941 gcagcctgaa ggggttgtgc tgggcaaagt gctcgtaaag gagattgaaa attcaggttt
     3001 cgagattaac gattctaaga ctagattgac ctacaaaaca agtaggcaag aagtcaccgg
     3061 gctgacggtt aatcggattg taaacattga tcggtgctac tacaaaaaga cgagggcgct
     3121 ggctcacgca ttgtatcgga caggagaata taaggtccca gacgagaacg gtgttctggt
     3181 atctggaggg cttgacaagt tggagggtat gtttgggttt atcgaccagg tggataaatt
     3241 caacaacatt aaaaaaaagt tgaataagca acccgacaga tatgttctga caaatgccac
     3301 tttgcacgga tttaagctca aattgaacgc cagggagaaa gcctatagca aattcatcta
     3361 ctacaaattc ttccacggta atacttgtcc cacgatcata acagagggta agacggatag
     3421 gatttacctt aaagctgccc tccatagcct cgagacaagt tatcctgaac tgtttcggga
     3481 gaaaacagat agtaagaaga aggagataaa tctgaatatt tttaaaagca atgagaagac
     3541 caagtatttc ctggatctca gcggcggcac agcagacctc aagaaattcg tggaacgcta
     3601 caaaaataac tacgcttcct attacggcag cgtaccgaaa caaccggtga taatggtgct
     3661 tgataacgac acaggcccgt cagacctgtt gaactttttg agaaacaaag ttaagagttg
     3721 tccagatgat gtaacagaaa tgcgcaagat gaagtacata catgtgtttt acaatctgta
     3781 catagttctg actcccctgt ctccatctgg agagcaaacg tctatggagg acctctttcc
     3841 taaagatata ttggacatta agatagatgg caagaaattc aataaaaaca atgacggtga
     3901 ctccaaaaca gagtatggga agcacatatt ctcaatgcgc gttgtacgag ataaaaagag
     3961 gaagatagat ttcaaggcat tttgctgtat cttcgatgct attaaggata ttaaagaaca
     4021 ttacaaactg atgttgaatt cctaggcggc cgcgggcatt taactatgat aagattccgt
     4081 acacgcatgt aggcagattt gttggttgtg aatcgcaacc agtggcctta atggcaggag
     4141 gaatcgcctc cctaaaatcc ttgattcaga gctatacggc aggtgtgctg tgcgaaggag
     4201 tgcctgcatg cgtgtacgga atcttatcaa ccggttagta atgagtttaa acaaagaaag
     4261 tggaatattc attcatatca tattttttct attaactgcc tggtttcttt taaatttttt
     4321 attggttgtc gacttgaacg gagtgacaat atatatatat atatttaata atgacatcat
     4381 tatctgtaaa tctgattctt aatgctattc tagttatgta agagtggtcc tttccataaa
     4441 aaaaaaaaaa gaaaaaagaa ttttaggaat acaatgcagc ttgtaagtaa aatctggaat
     4501 attcatatcg ccacaacttc ttatgcttat aaaagcacta atgcctgaat ttatgttgaa
     4561 aatatgtgtc acaaataaag aaactgtgac atctgacaca tttccacgta cccaattcgc
     4621 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     4681 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     4741 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     4801 aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4861 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     4921 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4981 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     5041 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     5101 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     5161 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     5221 atttaacgcg aattttaaca aaatattaac gcttacaatt tcctgatgcg gtattttctc
     5281 cttacgcatc tgtgcggtat ttcacaccgc atagggtaat aactgatata attaaattga
     5341 agctctaatt tgtgagttta gtatacatgc atttacttat aatacagttt tttagttttg
     5401 ctggccgcat cttctcaaat atgcttccca gcctgctttt ctgtaacgtt caccctctac
     5461 cttagcatcc cttccctttg caaatagtcc tcttccaaca ataataatgt cagatcctgt
     5521 agagaccaca tcatccacgg ttctatactg ttgacccaat gcgtctccct tgtcatctaa
     5581 acccacaccg ggtgtcataa tcaaccaatc gtaaccttca tctcttccac ccatgtctct
     5641 ttgagcaata aagccgataa caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt
     5701 agtatattct ccagtagata gggagccctt gcatgacaat tctgctaaca tcaaaaggcc
     5761 tctaggttcc tttgttactt cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc
     5821 caccacaccg tgtgcattcg taatgtctgc ccattctgct attctgtata cacccgcaga
     5881 gtactgcaat ttgactgtat taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa
     5941 attgtacttg gcggataatg cctttagcgg cttaactgtg ccctccatgg aaaaatcagt
     6001 caagatatcc acatgtgttt ttagtaaaca aattttggga cctaatgctt caactaactc
     6061 cagtaattcc ttggtggtac gaacatccaa tgaagcacac aagtttgttt gcttttcgtg
     6121 catgatatta aatagcttgg cagcaacagg actaggatga gtagcagcac gttccttata
     6181 tgtagctttc gacatgattt atcttcgttt cctgcaggtt tttgttctgt gcagttgggt
     6241 taagaatact gggcaatttc atgtttcttc aacactacat atgcgtatat ataccaatct
     6301 aagtctgtgc tccttccttc gttcttcctt ctgttcggag attaccgaat caaaaaaatt
     6361 tcaaggaaac cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga aaaggtggta
     6421 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     6481 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     6541 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     6601 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     6661 gtttcttaga cggatcgctt gcctgtaact tacacgcgcc tcgtatcttt taatgatgga
     6721 ataatttggg aatttactct gtgtttattt atttttatgt tttgtatttg gattttagaa
     6781 agtaaataaa gaaggtagaa gagttacgga atgaagaaaa aaaaataaac aaaggtttaa
     6841 aaaatttcaa caaaaagcgt actttacata tatatttatt agacaagaaa agcagattaa
     6901 atagatatac attcgattaa cgataagtaa aatgtaaaat cacaggattt tcgtgtgtgg
     6961 tcttctacac agacaagatg aaacaattcg gcattaatac ctgagagcag gaagagcaag
     7021 ataaaaggta gtatttgttg gcgatccccc tagagtcttt tacatcttcg gaaaacaaaa
     7081 actatt
//
