LOCUS       sequence-435231-        8324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Temperature sensitive replication (repA101ts) backbone encoding the 
            retron recombineering operon (ncRNA, RT, CspRecT, mutLE32K).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8324)
  AUTHORS   Gonzalez-Delgado A, Lopez SC, Rojas-Montero M, Fishman CB, Shipman 
            SL
  TITLE     Simultaneous multi-site editing of individual genomes using retron 
            arrays.
  JOURNAL   Nat Chem Biol. 2024 Nov;20(11):1482-1492. doi: 
            10.1038/s41589-024-01665-7. Epub 2024 Jul 9.
  PUBMED    38982310
REFERENCE   2  (bases 1 to 8324)
  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/s41589-024-01665-7"; journalName: "Nat Chem Biol"; date: 
            "2024-11"; volume: "20"; issue: "11"; pages: "1482-1492"
COMMENT     Sequence Label: pAGD248
FEATURES             Location/Qualifiers
     source          1..8324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(11..889)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        916..1200
                     /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     1123..1142
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1227..1235
                     /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)"
     RBS             1506..1528
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     RBS             2523..2531
                     /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      5288..5382
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5491..6441)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      6489..6711
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(7333..8124,8125..8193))
                     /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     7956..7975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8194..8298)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catcgattta ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac
       61 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaag tagagttgat
      121 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca
      181 gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct
      241 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga
      301 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat
      361 tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct
      421 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga
      481 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg
      541 tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt
      601 aaacccactg gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc
      661 ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca
      721 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt
      781 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg
      841 cattaaacga gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac
      901 tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg
      961 tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt
     1021 aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca
     1081 gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat
     1141 ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat
     1201 acccgttttt ttgggaattc gagctctaag gaggttataa aaaagattcc gtatgcgcac
     1261 ccttagcgag aggtttatca ttaaggtcaa cctctggatg ttgtttcggc atcctgcatt
     1321 gaatctgagt tactgtctgt tttcctgaat tcccgatttc cgcagcagtg aaagagttct
     1381 tcggttccag ccagctgcct cagtttatgg accagaacaa cccgctgtct gagattacgc
     1441 acgaattcag gaaacccgtt ttttctgacg taagggtgcg catacggaat ctagcggcaa
     1501 ataattttgt ttaactttaa gaaggagata tacatatgaa atccgctgaa tatttgaaca
     1561 cttttagatt gagaaatctc ggcctacctg tcatgaacaa tttgcatgac atgtctaagg
     1621 cgactcgcat atctgttgaa acacttcggt tgttaatcta tacagctgat tttcgctata
     1681 ggatctacac tgtagaaaag aaaggcccag agaagagaat gagaaccatt taccaacctt
     1741 ctcgagaact taaagcctta caaggatggg ttctacgtaa cattttagat aaactgtcgt
     1801 catctccttt ttctattgga tttgaaaagc accaatctat tttgaataat gctaccccgc
     1861 atattggggc aaactttata ctgaatattg atttggagga ttttttccca agtttaactg
     1921 ctaacaaagt ttttggagtg ttccattctc ttggttataa tcgactaata tcttcagttt
     1981 tgacaaaaat atgttgttat aaaaatctgc taccacaagg tgctccatca tcacctaaat
     2041 tagctaatct aatatgttct aaacttgatt atcgtattca gggttatgca ggtagtcggg
     2101 gcttgatata tacgagatat gccgatgatc tcaccttatc tgcacagtct atgaaaaagg
     2161 ttgttaaagc acgtgatttt ttattttcta taatcccaag tgaaggattg gttattaact
     2221 caaaaaaaac ttgtattagt gggcctcgta gtcagaggaa agttacaggt ttagttattt
     2281 cacaagagaa agttgggata ggtagagaaa aatataaaga aattagagca aagatacatc
     2341 atatattttg cggtaagtct tctgagatag aacacgttag gggatggttg tcatttattt
     2401 taagtgtgga ttcaaaaagc cataggagat taataactta tattagcaaa ttagaaaaaa
     2461 aatatggaaa gaacccttta aataaagcga agacctaaga tcgaaataat tttgtttaac
     2521 tttaaggagg tttggaatga accaaatcgt gaagttcact gacgactctg gcctggcggt
     2581 tcaagttact ccagacgatg ttcgccgtta tatctgtgag aacgctactg aaaaagaggt
     2641 gggcctcttt ctgcaactct gtcagactca acgtctgaat ccgtttgtga aagacgctta
     2701 cctggtgaaa tacggcggtg ctccagcttc tatgattact tcctatcaag tttttaaccg
     2761 tcgcgcgtgt cgtgatgcta actatgatgg tatcaaatct ggtgtggttg ttctgcgtga
     2821 cggtgatgtt gtgcataaac gtggtgctgc gtgctacaaa aaggcgggtg aggagctcat
     2881 cggtggttgg gcggaagttc gctttaagga tggccgcgag actgcgtatg ctgaggtggc
     2941 gctcgacgac tattccaccg gcaaatctaa ttgggcgaaa atgccgggtg ttatgatcga
     3001 aaaatgcgcg aaggctgctg cttggcgcct cgcgttcccg gacacttttc agggcatgta
     3061 cgctgcggag gaaatggatc aagcgcaaca gccagaacag gtgcgcgctc aggcggagca
     3121 accagtggat ctccagccaa tccgcgaact cttcaagcca tattgcgaac acttcggcat
     3181 cactccggct gagggtatga ctgctgtttg tggtgcggtg ggcgctgaag gcatgcactc
     3241 tatgaccgag cagcaagctc gccgtgctcg cgcttggatg gaggaagaaa tggctgcgcc
     3301 agctgtggaa gcggagtatg aggttgttga cgagggcgag gtgttttaag gatccaggag
     3361 aatacacatg ccaattcagg tcttaccgcc acaactggcg aaccagattg ccgcaggtga
     3421 ggtggtcgag cgacctgcgt cggtagtcaa agaactagtg aaaaacagcc tcgatgcagg
     3481 tgcgacgcgt atcgatattg atatcgaacg cggtggggcg aaacttatcc gcattcgtga
     3541 taacggctgc ggtatcaaaa aagatgagct ggcgctggcg ctggctcgtc atgccaccag
     3601 taaaatcgcc tctctggacg atctcgaagc cattatcagc ctgggctttc gcggtgaggc
     3661 gctggcgagt atcagttcgg tttcccgcct gacgctcact tcacgcaccg cagaacagca
     3721 ggaagcctgg caggcctatg ccgaagggcg cgatatgaac gtgacggtaa aaccggcggc
     3781 gcatcctgtg gggacgacgc tggaggtgct ggatctgttc tacaacaccc cggcgcggcg
     3841 caaattcctg cgcaccgaga aaaccgaatt taaccacatt gatgagatca tccgccgcat
     3901 tgcgctggcg cgtttcgacg tcacgatcaa cctgtcgcat aacggtaaaa ttgtgcgtca
     3961 gtaccgcgca gtgccggaag gcgggcaaaa agaacggcgc ttaggcgcga tttgcggcac
     4021 cgcttttctt gaacaagcgc tggcgattga atggcaacac ggcgatctca cgctacgcgg
     4081 ctgggtggcc gatccaaatc acaccacgcc cgcactggca gaaattcagt attgctacgt
     4141 gaacggtcgc atgatgcgcg atcgcctgat caatcacgcg atccgccagg cctgcgaaga
     4201 caaactgggg gccgatcagc aaccggcatt tgtgttgtat ctggagatcg acccacatca
     4261 ggtggacgtc aacgtgcacc ccgccaaaca cgaagtgcgt ttccatcagt cgcgtctggt
     4321 gcatgatttt atctatcagg gcgtgctgag cgtgctacaa cagcaactgg aaacgccgct
     4381 accgctggac gatgaacccc aacctgcacc gcgttccatt ccggaaaacc gcgtggcggc
     4441 ggggcgcaat cactttgcag aaccggcagc tcgtgagccg gtagctccgc gctacactcc
     4501 tgcgccagca tcaggcagtc gtccggctgc cccctggccg aatgcgcagc caggctacca
     4561 gaaacagcaa ggtgaagtgt atcgccagct tttgcaaacg cccgcgccga tgcaaaaatt
     4621 aaaagcgccg gaaccgcagg aacctgcact tgcggcgaac agtcagagtt ttggtcgggt
     4681 actgactatc gtccattccg actgtgcgtt gctggagcgc gacggcaaca tttcactttt
     4741 atccttgcca gtggcagaac gttggctgcg tcaggcacaa ttgacgccgg gtgaagcgcc
     4801 cgtttgcgcc cagccgctgc tgattccgtt gcggctaaaa gtttctgccg aagaaaaatc
     4861 ggcattagaa aaagcgcagt ctgccctggc ggaattgggt attgatttcc agtcagatgc
     4921 acagcatgtg accatcaggg cagtgccttt acccttacgc caacaaaatt tacaaatctt
     4981 gattcctgaa ctgataggct acctggcgaa gcagtccgta ttcgaacctg gcaatattgc
     5041 gcagtggatt gcacgaaatc tgatgagcga acatgcgcag tggtcaatgg cacaggccat
     5101 aaccctgctg gcggacgtgg aacggttatg tccgcaactt gtgaaaacgc cgccgggtgg
     5161 tctgttacaa tctgttgatt tacatccggc gataaaagcc ctgaaagatg agtgatctag
     5221 agtcgacctg caggcatgca agcttgcggc cgcgtcgtga ctgggaaaac cctggcgact
     5281 agtcttggac tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc
     5341 agaacgctcg gttgccgccg ggcgtttttt attggtgaga attccatggg tatggacagt
     5401 tttccctttg atatgtaacg gtgaacagtt gttctacttt tgtttgttag tcttgatgct
     5461 tcactgatag atacaagagc cataagaacc tcagatcctt ccgtatttag ccagtatgtt
     5521 ctctagtgtg gttcgttgtt tttgcgtgag ccatgagaac gaaccattga gatcatactt
     5581 actttgcatg tcactcaaaa attttgcctc aaaactggtg agctgaattt ttgcagttaa
     5641 agcatcgtgt agtgtttttc ttagtccgtt atgtaggtag gaatctgatg taatggttgt
     5701 tggtattttg tcaccattca tttttatctg gttgttctca agttcggtta cgagatccat
     5761 ttgtctatct agttcaactt ggaaaatcaa cgtatcagtc gggcggcctc gcttatcaac
     5821 caccaatttc atattgctgt aagtgtttaa atctttactt attggtttca aaacccattg
     5881 gttaagcctt ttaaactcat ggtagttatt ttcaagcatt aacatgaact taaattcatc
     5941 aaggctaatc tctatatttg ccttgtgagt tttcttttgt gttagttctt ttaataacca
     6001 ctcataaatc ctcatagagt atttgttttc aaaagactta acatgttcca gattatattt
     6061 tatgaatttt tttaactgga aaagataagg caatatctct tcactaaaaa ctaattctaa
     6121 tttttcgctt gagaacttgg catagtttgt ccactggaaa atctcaaagc ctttaaccaa
     6181 aggattcctg atttccacag ttctcgtcat cagctctctg gttgctttag ctaatacacc
     6241 ataagcattt tccctactga tgttcatcat ctgaacgtat tggttataag tgaacgatac
     6301 cgtccgttct ttccttgtag ggttttcaat cgtggggttg agtagtgcca cacagcataa
     6361 aattagcttg gtttcatgct ccgttaagtc atagcgacta atcgctagtt catttgcttt
     6421 gaaaacaact aattcagaca tacatctcaa ttggtctagg tgattttaat cactatacca
     6481 attgagatgg gctagtcaat gataattact agtccttttc ctttgagttg tgggtatctg
     6541 taaattctgc tagacctttg ctggaaaact tgtaaattct gctagaccct ctgtaaattc
     6601 cgctagacct ttgtgtgttt tttttgttta tattcaagtg gttataattt atagaataaa
     6661 gaaagaataa aaaaagataa aaagaataga tcccagccct gtgtataact cactacttta
     6721 gtcagttccg cagtattaca aaaggatgtc gcaaacgctg tttgctcctc tacaaaacag
     6781 accttaaaac cctaaaggct taagtagcac cctcgcaagc tcggttgcgg ccgcaatcgg
     6841 gcaaatcgct gaatattcct tttgtctccg accatcaggc acctgagtcg ctgtcttttt
     6901 cgtgacattc agttcgctgc gctcacggct ctggcagtga atgggggtaa atggcactac
     6961 aggcgccttt tatggattca tgcaaggaaa ctacccataa tacaagaaaa gcccgtcacg
     7021 ggcttctcag ggcgttttat ggcgggtctg ctatgtggtg ctatctgact ttttgctgtt
     7081 cagcagttcc tgccctctga ttttccagtc tgaccacttc ggattatccc gtgacaggtc
     7141 attcagactg gctaatgcac ccagtaaggc agcggtatca tcaacggggt ctgacgctca
     7201 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     7261 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     7321 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     7381 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     7441 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     7501 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7561 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7621 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7681 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7741 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7801 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7861 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7921 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7981 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     8041 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     8101 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     8161 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     8221 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     8281 acaaataggg gttccgcgca catttccccg aaaagtgcca cctg
//
