LOCUS       sequence-355782-        6802 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses retron RNA, ec.86 Reverse Transcriptase, and CspRecT SSAP,
            to create a specific rifampicin resistance mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6802)
  AUTHORS   Schubert MG, Goodman DB, Wannier TM, Kaur D, Farzadfard F, Lu TK, 
            Shipman SL, Church GM
  TITLE     High-throughput functional variant screens via in vivo production of
            single-stranded DNA.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 May 4;118(18). pii: 2018181118. doi: 
            10.1073/pnas.2018181118.
  PUBMED    33906944
REFERENCE   2  (bases 1 to 6802)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2021-05-4"; volume: "118(18). pii: 
            2018181118. doi"; pages: " 10.1073/pnas.2018181118"
COMMENT     Sequence Label: pMS_366
FEATURES             Location/Qualifiers
     source          1..6802
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(86..964)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        991..1275
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1198..1217
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             2513..2521
                     /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)"
     primer_bind     complement(3402..3419)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(4165..5115)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      5163..5385
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(5877..5971)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6016..6675)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6590..6609
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6676..6778)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac tccgtcaagc
       61 cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca ttcacttttt
      121 cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg
      181 agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg
      241 tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc
      301 taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct
      361 gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg tactgacaag
      421 cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc
      481 gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt
      541 gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg
      601 ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg
      661 cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt
      721 agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc
      781 gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca
      841 ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac
      901 ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt tgcgcttcag
      961 ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat tgcatcagac
     1021 attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta accccgctta
     1081 ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt aacaaaagtg
     1141 tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca ctttgctatg
     1201 ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta tcgcaactct
     1261 ctactgtttc tccatacccg tttaattaaa tgcgcaccct tagcgagagg tttatcatta
     1321 aggtcaacct ctggatgttg tttcggcatc ctgcattgaa tctgagttac tgtctgtttt
     1381 cctgaattcc agtgaaagag ttcttcggtt ccagtcagct gtctcagttt atgggccaga
     1441 acaacccgct gtctgagatt acgcacaaac gtcgtatctg aattcaggaa acccgttttt
     1501 tctgacgtaa gggtgcgcaa ctttcatgaa atccgctgaa tatttgaaca cttttagatt
     1561 gagaaatctc ggcctacctg tcatgaacaa tttgcatgac atgtctaagg cgactcgcat
     1621 atctgttgaa acacttcggt tgttaatcta tacagctgat tttcgctata ggatctacac
     1681 tgtagaaaag aaaggcccag agaagagaat gagaaccatt taccaacctt ctcgagaact
     1741 taaagcctta caaggatggg ttctacgtaa cattttagat aaactgtcgt catctccttt
     1801 ttctattgga tttgaaaagc accaatctat tttgaataat gctaccccgc atattggggc
     1861 aaactttata ctgaatattg atttggagga ttttttccca agtttaactg ctaacaaagt
     1921 ttttggagtg ttccattctc ttggttataa tcgactaata tcttcagttt tgacaaaaat
     1981 atgttgttat aaaaatctgc taccacaagg tgctccatca tcacctaaat tagctaatct
     2041 aatatgttct aaacttgatt atcgtattca gggttatgca ggtagtcggg gcttgatata
     2101 tacgagatat gccgatgacc tcaccttatc tgcacagtct atgaaaaagg ttgttaaagc
     2161 acgtgatttt ttattttcta taatcccaag tgaaggattg gttattaact caaaaaaaac
     2221 ttgtattagt gggcctcgta gtcagaggaa agttacaggt ttagttattt cacaagagaa
     2281 agttgggata ggtagagaaa aatataaaga aattagagca aagatacatc atatattttg
     2341 cggtaagtct tctgagatag aacacgttag gggatggttg tcatttattt taagtgtgga
     2401 ttcaaaaagc cataggagat taataactta tattagcaaa ttagaaaaaa aatatggaaa
     2461 gaacccttta aataaagcga agacctagga tcgaaataat tttgtttaac tttaaggagg
     2521 tttggaatga accaaatcgt gaagttcact gacgactctg gcctggcggt tcaagttact
     2581 ccagacgatg ttcgccgtta tatctgtgag aacgctactg aaaaagaggt gggcctcttt
     2641 ctgcaactct gtcagactca acgtctgaat ccgtttgtga aagacgctta cctggtgaaa
     2701 tacggcggtg ctccagcttc tatgattact tcctatcaag tttttaaccg tcgcgcgtgt
     2761 cgtgatgcta actatgatgg tatcaaatct ggtgtggttg ttctgcgtga cggtgatgtt
     2821 gtgcataaac gtggtgctgc gtgctacaaa aaggcgggtg aggagctcat cggtggttgg
     2881 gcggaagttc gctttaagga tggccgcgag actgcgtatg ctgaggtggc gctcgacgac
     2941 tattccaccg gcaaatctaa ttgggcgaaa atgccgggtg ttatgatcga aaaatgcgcg
     3001 aaggctgctg cttggcgcct cgcgttcccg gacacttttc agggcatgta cgctgcggag
     3061 gaaatggatc aagcgcaaca gccagaacag gtgcgcgctc aggcggagca accagtggat
     3121 ctccagccaa tccgcgaact cttcaagcca tattgcgaac acttcggcat cactccggct
     3181 gagggtatga ctgctgtttg tggtgcggtg ggcgctgaag gcatgcactc tatgaccgag
     3241 cagcaagctc gccgtgctcg cgcttggatg gaggaagaaa tggctgcgcc agctgtggaa
     3301 gcggagtatg aggttgttga cgagggcgag gtgttttaat ctagagtcga cctgcaggca
     3361 tgcaagcttg gctgttttgg cggatgagag aagattttca gcctgataca gattaaatca
     3421 gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca
     3481 cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct
     3541 ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     3601 ctgaacgctc tcctgagtag gacaaatccg ccgccctaga cctagggtac gggttttgct
     3661 gcccgcaaac gggctgttct ggtgttgcta gtttgttatc agaatcgcag atccggcttc
     3721 agccggtttg ccggctgaaa gcgctatttc ttccagaatt gccatgattt tttccccacg
     3781 ggaggcgtca ctggctcccg tgttgtcggc agctttgatt cgataagcag catcgcctgt
     3841 ttcaggctgt ctatgtgtga ctgttgagct gtaacaagtt gtctcaggtg ttcaatttca
     3901 tgttctagtt gctttgtttt actggtttca cccgttctat taggtgttac atgctgttca
     3961 tctgttacat tgtcgatctg ttcatggtga acagctttga atgcaccaaa aactcgtaaa
     4021 agctctgatg tatctatctt ttttacaccg ttttcatctg tgcatatgga cagttttccc
     4081 tttgatatgt aacggtgaac agttgttcta cttttgtttg ttagtcttga tgcttcactg
     4141 atagatacaa gagccataag aacctcagat ccttccgtat ttagccagta tgttctctag
     4201 tgtggttcgt tgtttttgcg tgagccatga gaacgaacca ttgagatcat acttactttg
     4261 catgtcactc aaaaattttg cctcaaaact ggtgagctga atttttgcag ttaaagcatc
     4321 gtgtagtgtt tttcttagtc cgttatgtag gtaggaatct gatgtaatgg ttgttggtat
     4381 tttgtcacca ttcattttta tctggttgtt ctcaagttcg gttacgagat ccatttgtct
     4441 atctagttca acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat caaccaccaa
     4501 tttcatattg ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc attggttaag
     4561 ccttttaaac tcatggtagt tattttcaag cattaacatg aacttaaatt catcaaggct
     4621 aatctctata tttgccttgt gagttttctt ttgtgttagt tcttttaata accactcata
     4681 aatcctcata gagtatttgt tttcaaaaga cttaacatgt tccagattat attttatgaa
     4741 tttttttaac tggaaaagat aaggcaatat ctcttcacta aaaactaatt ctaatttttc
     4801 gcttgagaac ttggcatagt ttgtccactg gaaaatctca aagcctttaa ccaaaggatt
     4861 cctgatttcc acagttctcg tcatcagctc tctggttgct ttagctaata caccataagc
     4921 attttcccta ctgatgttca tcatctgagc gtattggtta taagtgaacg ataccgtccg
     4981 ttctttcctt gtagggtttt caatcgtggg gttgagtagt gccacacagc ataaaattag
     5041 cttggtttca tgctccgtta agtcatagcg actaatcgct agttcatttg ctttgaaaac
     5101 aactaattca gacatacatc tcaattggtc taggtgattt taatcactat accaattgag
     5161 atgggctagt caatgataat tactagtcct tttcctttga gttgtgggta tctgtaaatt
     5221 ctgctagacc tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa attccgctag
     5281 acctttgtgt gttttttttg tttatattca agtggttata atttatagaa taaagaaaga
     5341 ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat aactcactac tttagtcagt
     5401 tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa acagacctta
     5461 aaaccctaaa ggcttaagta gcaccctcgc aagctcgggc aaatcgctga atattccttt
     5521 tgtctccgac catcaggcac ctgagtcgct gtctttttcg tgacattcag ttcgctgcgc
     5581 tcacggctct ggcagtgaat gggggtaaat ggcactacag gcgcctttta tggattcatg
     5641 caaggaaact acccataata caagaaaagc ccgtcacggg cttctcaggg cgttttatgg
     5701 cgggtctgct atgtggtgct atctgacttt ttgctgttca gcagttcctg ccctctgatt
     5761 ttccagtctg accacttcgg attatcccgt gacaggtcat tcagactggc taatgcaccc
     5821 agtaaggcag cggtatcatc aacaggctta cccgtcttac tgtccctagt gcttggattc
     5881 tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct
     5941 gaggtcatta ctggatctat caacaggagt ccaagcgagc tcgatatccg tcggcttgaa
     6001 cgaattgtta gacaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt
     6061 aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg
     6121 catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa
     6181 gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga
     6241 gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca
     6301 cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca
     6361 gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc
     6421 ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag cattcatcag
     6481 gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt
     6541 taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg
     6601 aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt
     6661 gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac
     6721 gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac
     6781 gtctcatttt cgccagatat cg
//
