LOCUS       sequence-367502-        5682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses retron Eco4 RT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5682)
  AUTHORS   Bhattarai-Kline S, Lear SK, Fishman CB, Lopez SC, Lockshin ER, 
            Schubert MG, Nivala J, Church GM, Shipman SL
  TITLE     Recording gene expression order in DNA by CRISPR addition of retron 
            barcodes.
  JOURNAL   Nature. 2022 Aug;608(7921):217-225. doi: 10.1038/s41586-022-04994-6.
            Epub 2022 Jul 27.
  PUBMED    35896746
REFERENCE   2  (bases 1 to 5682)
  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/s41586-022-04994-6"; journalName: "Nature"; date: 
            "2022-08"; volume: "608"; issue: "7921"; pages: "217-225"
COMMENT     Sequence Label: pSLS.400
FEATURES             Location/Qualifiers
     source          1..5682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(118..135)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(252..797)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(332..351)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1035..1826)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVITEVSTQLSEVVGAIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     primer_bind     complement(2221..2238)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2391..2477
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             3861..3869
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     terminator      complement(4476..4570)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        complement(5343..5447)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5515..5533
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      5601..5648
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tcagatcctt ccgtatttag ccagtatgtt ctctagacgg atcgcttcat gtggtaggag
       61 aaaaaaggct gcaccggtgc gtcagcagaa tatgtgatac aggatatatt ccgcttcctc
      121 gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg cttacgaacg
      181 gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg
      241 gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca
      301 aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggcggc
      361 tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc gctgttatgg
      421 ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc gctccaagct
      481 ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg
      541 tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca ctggtaattg
      601 atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt
      661 ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta gctcagagaa
      721 ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag attacgcgca
      781 gaccaaaacg atctcaagaa gatcatctta ttaaggggtc tgacgctcag tggaacgaaa
      841 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccagc
      901 ggtatcatca acaggcttac ccgtcttact gtcaagagga catccggtca aataaaacga
      961 aaggctcagt cgaaagactg ggcctttcgt tttgctgagg agacttaggg acccttcgaa
     1021 ccccagagtc ccgcttattt gccgactacc ttggtgatct cgcctttcac gtagtggaca
     1081 aattcttcca actgatctgc gcgcgaggcc aagcgatctt cttcttgtcc aagataagcc
     1141 tgtctagctt caagtatgac gggctgatat tgggccggca ggcgctccat tgcccagtcg
     1201 gcagcgacat ccttcggcgc gattttgccg gttactgcgc tgtaccaaat gcgggacaac
     1261 gtaagcacta catttcgctc atcgccagcc cagtcgggcg gcgagttcca tagcgttaag
     1321 gtttcattta gcgcctcaaa tagatcctgt tcaggaaccg gatcaaagag ttcctccgcc
     1381 gctggaccta ccaaggcaac gctatgttct cttgcttttg tcagcaagat agccagatca
     1441 atgtcgatcg tggctggctc gaagatacct gcaagaatgt cattgcgctg ccattctcca
     1501 aattgcagtt cgcgcttagc tggataacgc cacggaatga tgtcgtcgtg cacaacaatg
     1561 gtgacttcta cagcgcggag aatctcgctc tctccagggg aagccgaagt ttccaaaagg
     1621 tcgttgatca aagctcgccg cgttgtttca tcaagcctta cggtcaccgt aactagcaaa
     1681 tcaatatcac tgtgtggctt caggccgcca tccactgcgg agccgtacaa atgtacggcc
     1741 agcaacgtcg gttcgagatg gcgctcgatg gcgccaacta cctctgatag ttgagtcgat
     1801 acttcggtga tcaccgcttc cctcataatg tttaactttg ttttagggcg actgccctgc
     1861 tgcgtaacat cgttgctgct ccataacatc aaacatcgac ccatggcgta acgcgcttgc
     1921 tgcttggatg cccgaggcat agactgtacc ccaaaaaaac agtcatagca agccatgaaa
     1981 accgccactg caccgttacc accgctgcgt tcggtcaagg ttctggacca gttgcgtgag
     2041 cgcatgcgaa acgatcctca tcctgtctct tgatcagatc ttgatcccct gcgccatcag
     2101 atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac cttaccagag
     2161 ggcgccccag ctggcaattc cgacgtctgg ctgttttggc ggatgagaga agattttcag
     2221 cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg
     2281 cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc
     2341 cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac
     2401 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     2461 tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag
     2521 ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag aaggccatcc
     2581 gtcaggatgg cctttttgcg tttctacaaa ctggattgaa tataaccgac gtgactgtta
     2641 catttaggtg gctaaacccg tcaacattta actttaagaa ggagatatac atatgagcat
     2701 cgacattgaa acgacacttc aaaaagctta ccccgatttt gacgtacttt taaaatcaag
     2761 accagcgaca cactacaaag tatacaaaat tccaaagcgt acaattggct atcgtatcat
     2821 agctcaacct acgccacgag taaaagcaat acaaagggat ataattgaaa tactcaaaca
     2881 gcatactcat atccacgatg cagccacagc gtatgttgat gggaagaata tattagataa
     2941 cgccaagata catcaaagct cagtttattt attaaagcta gatttggtca atttttttaa
     3001 taaaataaca ccggaacttt tatttaaggc attagctaga caaaaagtag atatttcaga
     3061 taccaacaaa aatttattaa aacaattttg tttttggaat agaacaaaaa gaaaaaatgg
     3121 tgcgttagtt ttaagtgtag gagcaccaag ctctcctttt atatcgaata ttgttatgag
     3181 cagcttcgat gaagagataa gttctttttg caaggaaaat aaaatatctt actcaagata
     3241 tgctgacgac ttaacttttt caacaaatga aagagatgtt ttagggttag ctcatcaaaa
     3301 agtaaaaaca acattaattc gtttttttgg cacgcggatt attattaata ataacaagat
     3361 cgtatattct tctaaagcac ataatcgcca tgtcacagga gtgacgttaa ctaataataa
     3421 taaattatca ctaggaagag aaaggaaaag atatattact tctttagtat ttaaatttaa
     3481 agaaggcaaa ctttcgaatg tagatattaa tcatcttaga ggcttgatcg gttttgctta
     3541 taatattgag ccagctttta ttgaaagact tgagaaaaaa tatggagaat caaccattaa
     3601 gtctatcaag aaatactctg aaggaggata attaattaac ggcactcctc agcaaatata
     3661 atgaccctct tgataaccca agagggcatt ttttaatgcc catggcgttt accacagcta
     3721 acaccacgtc gtccctatct gctgccctag gtctatgagt ggttgctgga taactttacg
     3781 ggcatgcata aggctcgtag gctatattca gggagaccac aacggtttcc ctctacaaat
     3841 aattttgttt aactttgaaa taaggaggta atacaaatgc cgcgtccgaa actgaaatct
     3901 gacgacgaag ttctggaagc ggcgaccgtt gttctgaaac gttgcggtcc gatcgaattc
     3961 accctgtctg gtgttgcgaa agaagttggt ctgtctcgtg cggcgctgat ccagcgtttc
     4021 accaaccgtg acaccctgct ggttcgtatg atggaacgtg gtgttgaaca ggttcgtcac
     4081 tacctgaacg cgatcccgat cggtgcgggt ccgcagggtc tgtgggaatt cctgcaggtt
     4141 ctggttcgtt ctatgaacac ccgtaacgac ttctctgtta actacctgat ctcttggtac
     4201 gaactgcagg ttccggaact gcgtaccctg gcgatccagc gtaaccgtgc ggttgttgaa
     4261 ggtatccgta aacgtctgcc gccgggtgcg ccggcggcgg cggaactgct gctgcactct
     4321 gttatcgcgg gtgcgaccat gcagtgggcg gttgacccgg acggtgaact ggcggaccac
     4381 gttctggcgc agatcgcggc gatcctgtgc ctgatgttcc cggaacacga cgacttccag
     4441 ctgctgcagc cgcacgcgta ataactagtg cttggattct caccaataaa aaacgcccgg
     4501 cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc
     4561 aacaggagtc caagcgagct cgtaaacttg gtctgacagc ccttaactta cttattaaat
     4621 aatttatagc tattgaaaag agataagaat tgttcaaagc taatattgtt taaatcgtca
     4681 attcctgcat gttttaagga attgttaaat tgattttttg taaatatttt cttgtattct
     4741 ttgttaaccc atttcataac gaaataatta tacttttgtt tatctttgtg tgatattctt
     4801 gatttttttc tacttaatct gataagtgag ctattcactt taggtttagg atgaaaatat
     4861 tctcttggaa ccatacttaa tatagaaata tcaacttctg ccattaaaag taatgccaat
     4921 gagcgttttg tatttaataa tcttttagca aacccgtatt ccacgattaa ataaatctca
     4981 ttagctatac tatcaaaaac aattttgcgt attatatccg tacttatgtt ataaggtata
     5041 ttaccatata ttttatagga ttggttttta ggaaatttaa actgcaatat atccttgttt
     5101 aaaacttgga aattatcgtg atcaacaagt ttattttctg tagttttgca taatttatgg
     5161 tctatttcaa tggcagttac gaaattacac ctctttacta attcaagggt aaaatggcct
     5221 tttcctgagc cgatttcaaa gatattatca tgttcattta atcttatatt tgtcattatt
     5281 ttatctatat tatgttttga agtaataaag ttttgactgt gttttatatt tttctcgttc
     5341 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5401 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5461 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     5521 cgtatcacga ggccctttcg tcttcactaa gcgactaaaa aattgaatgt aggaaaccaa
     5581 catgccagtt ctagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     5641 gttttttgct gaaacctcag gcgagaagca cacggtcaca ct
//
