LOCUS       sequence-355786-        8556 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses retron RNA, ec.86 Reverse Transcriptase, CspRecT SSAP, and
            mutL* to create a specific rifampicin resistance mutation while 
            inhibiting MMR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8556)
  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 8556)
  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_375
FEATURES             Location/Qualifiers
     source          1..8556
                     /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)"
     misc_feature    5341..5359
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     CDS             complement(5919..6869)
                     /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      6917..7139
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(7631..7725)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7770..8429)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     8344..8363
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(8430..8532)
                     /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 gtgttttaaa tggcgatgag agaggaggat
     3361 tacaaaatgc caattcaggt cttaccgcca caactggcga accagattgc cgcaggtgag
     3421 gtggtcgagc gacctgcgtc ggtagtcaaa gaactagtga aaaacagcct cgatgcaggt
     3481 gcgacgcgta tcgatattga tatcgaacgc ggtggggcga aacttatccg cattcgtgat
     3541 aacggctgcg gtatcaaaaa agatgagctg gcgctggcgc tggctcgtca tgccaccagt
     3601 aaaatcgcct ctctggacga tctcgaagcc attatcagcc tgggctttcg cggtgaggcg
     3661 ctggcgagta tcagttcggt ttcccgcctg acgctcactt cacgcaccgc agaacagcag
     3721 gaagcctggc aggcctatgc cgaagggcgc gatatgaacg tgacggtaaa accggcggcg
     3781 catcctgtgg ggacgacgct ggaggtgctg gatctgttct acaacacccc ggcgcggcgc
     3841 aaattcctgc gcaccgagaa aaccgaattt aaccacattg atgagatcat ccgccgcatt
     3901 gcgctggcgc gtttcgacgt cacgatcaac ctgtcgcata acggtaaaat tgtgcgtcag
     3961 taccgcgcag tgccggaagg cgggcaaaaa gaacggcgct taggcgcgat ttgcggcacc
     4021 gcttttcttg aacaagcgct ggcgattgaa tggcaacacg gcgatctcac gctacgcggc
     4081 tgggtggccg atccaaatca caccacgccc gcactggcag aaattcagta ttgctacgtg
     4141 aacggtcgca tgatgcgcga tcgcctgatc aatcacgcga tccgccaggc ctgcgaagac
     4201 aaactggggg ccgatcagca accggcattt gtgttgtatc tggagatcga cccacatcag
     4261 gtggacgtca acgtgcaccc cgccaaacac gaagtgcgtt tccatcagtc gcgtctggtg
     4321 catgatttta tctatcaggg cgtgctgagc gtgctacaac agcaactgga aacgccgcta
     4381 ccgctggacg atgaacccca acctgcaccg cgttccattc cggaaaaccg cgtggcggcg
     4441 gggcgcaatc actttgcaga accggcagct cgtgagccgg tagctccgcg ctacactcct
     4501 gcgccagcat caggcagtcg tccggctgcc ccctggccga atgcgcagcc aggctaccag
     4561 aaacagcaag gtgaagtgta tcgccagctt ttgcaaacgc ccgcgccgat gcaaaaatta
     4621 aaagcgccgg aaccgcagga acctgcactt gcggcgaaca gtcagagttt tggtcgggta
     4681 ctgactatcg tccattccga ctgtgcgttg ctggagcgcg acggcaacat ttcactttta
     4741 tccttgccag tggcagaacg ttggctgcgt caggcacaat tgacgccggg tgaagcgccc
     4801 gtttgcgccc agccgctgct gattccgttg cggctaaaag tttctgccga agaaaaatcg
     4861 gcattagaaa aagcgcagtc tgccctggcg gaattgggta ttgatttcca gtcagatgca
     4921 cagcatgtga ccatcagggc agtgccttta cccttacgcc aacaaaattt acaaatcttg
     4981 attcctgaac tgataggcta cctggcgaag cagtccgtat tcgaacctgg caatattgcg
     5041 cagtggattg cacgaaatct gatgagcgaa catgcgcagt ggtcaatggc acaggccata
     5101 accctgctgg cggacgtgga acggttatgt ccgcaacttg tgaaaacgcc gccgggtggt
     5161 ctgttacaat ctgttgattt acatccggcg ataaaagccc tgaaagatga gtgaaatggc
     5221 gatgagcatc ctcacgataa tatccgggta ggcgcaatca ctttcgtcta ctccgttaca
     5281 aagcgaggct gggtatttcc cggcctttct gttatccgaa atccactgaa agcacagcgg
     5341 agatgtgtat aagagacagc tgcctcctga gtaggacaaa tccgccgccc tagacctagg
     5401 gtacgggttt tgctgcccgc aaacgggctg ttctggtgtt gctagtttgt tatcagaatc
     5461 gcagatccgg cttcagccgg tttgccggct gaaagcgcta tttcttccag aattgccatg
     5521 attttttccc cacgggaggc gtcactggct cccgtgttgt cggcagcttt gattcgataa
     5581 gcagcatcgc ctgtttcagg ctgtctatgt gtgactgttg agctgtaaca agttgtctca
     5641 ggtgttcaat ttcatgttct agttgctttg ttttactggt ttcacccgtt ctattaggtg
     5701 ttacatgctg ttcatctgtt acattgtcga tctgttcatg gtgaacagct ttgaatgcac
     5761 caaaaactcg taaaagctct gatgtatcta tcttttttac accgttttca tctgtgcata
     5821 tggacagttt tccctttgat atgtaacggt gaacagttgt tctacttttg tttgttagtc
     5881 ttgatgcttc actgatagat acaagagcca taagaacctc agatccttcc gtatttagcc
     5941 agtatgttct ctagtgtggt tcgttgtttt tgcgtgagcc atgagaacga accattgaga
     6001 tcatacttac tttgcatgtc actcaaaaat tttgcctcaa aactggtgag ctgaattttt
     6061 gcagttaaag catcgtgtag tgtttttctt agtccgttat gtaggtagga atctgatgta
     6121 atggttgttg gtattttgtc accattcatt tttatctggt tgttctcaag ttcggttacg
     6181 agatccattt gtctatctag ttcaacttgg aaaatcaacg tatcagtcgg gcggcctcgc
     6241 ttatcaacca ccaatttcat attgctgtaa gtgtttaaat ctttacttat tggtttcaaa
     6301 acccattggt taagcctttt aaactcatgg tagttatttt caagcattaa catgaactta
     6361 aattcatcaa ggctaatctc tatatttgcc ttgtgagttt tcttttgtgt tagttctttt
     6421 aataaccact cataaatcct catagagtat ttgttttcaa aagacttaac atgttccaga
     6481 ttatatttta tgaatttttt taactggaaa agataaggca atatctcttc actaaaaact
     6541 aattctaatt tttcgcttga gaacttggca tagtttgtcc actggaaaat ctcaaagcct
     6601 ttaaccaaag gattcctgat ttccacagtt ctcgtcatca gctctctggt tgctttagct
     6661 aatacaccat aagcattttc cctactgatg ttcatcatct gagcgtattg gttataagtg
     6721 aacgataccg tccgttcttt ccttgtaggg ttttcaatcg tggggttgag tagtgccaca
     6781 cagcataaaa ttagcttggt ttcatgctcc gttaagtcat agcgactaat cgctagttca
     6841 tttgctttga aaacaactaa ttcagacata catctcaatt ggtctaggtg attttaatca
     6901 ctataccaat tgagatgggc tagtcaatga taattactag tccttttcct ttgagttgtg
     6961 ggtatctgta aattctgcta gacctttgct ggaaaacttg taaattctgc tagaccctct
     7021 gtaaattccg ctagaccttt gtgtgttttt tttgtttata ttcaagtggt tataatttat
     7081 agaataaaga aagaataaaa aaagataaaa agaatagatc ccagccctgt gtataactca
     7141 ctactttagt cagttccgca gtattacaaa aggatgtcgc aaacgctgtt tgctcctcta
     7201 caaaacagac cttaaaaccc taaaggctta agtagcaccc tcgcaagctc gggcaaatcg
     7261 ctgaatattc cttttgtctc cgaccatcag gcacctgagt cgctgtcttt ttcgtgacat
     7321 tcagttcgct gcgctcacgg ctctggcagt gaatgggggt aaatggcact acaggcgcct
     7381 tttatggatt catgcaagga aactacccat aatacaagaa aagcccgtca cgggcttctc
     7441 agggcgtttt atggcgggtc tgctatgtgg tgctatctga ctttttgctg ttcagcagtt
     7501 cctgccctct gattttccag tctgaccact tcggattatc ccgtgacagg tcattcagac
     7561 tggctaatgc acccagtaag gcagcggtat catcaacagg cttacccgtc ttactgtccc
     7621 tagtgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt ctgaacaaat
     7681 ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc gagctcgata
     7741 tccgtcggct tgaacgaatt gttagacaat tacgccccgc cctgccactc atcgcagtac
     7801 tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc
     7861 tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa
     7921 acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc
     7981 cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg
     8041 ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg
     8101 tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa
     8161 gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg gaattccgga
     8221 tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt
     8281 ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat
     8341 tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg
     8401 gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgat
     8461 aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt
     8521 acgtgccgat caacgtctca ttttcgccag atatcg
//
