LOCUS       sequence-331328-        5984 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Generates ssDNA in vivo targeting E. coli rpoB for recombineering w/
            Beta recombinase; temp. sensitive pSC101 ori and sgRNA for Cas9 
            counterselection; aada for spectinomycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5984)
  AUTHORS   Ellington AJ, Reisch CR
  TITLE     Efficient and iterative retron-mediated in vivo recombineering in 
            Escherichia coli.
  JOURNAL   Synth Biol (Oxf). 2022 May 3;7(1):ysac007. doi: 
            10.1093/synbio/ysac007. eCollection 2022.
  PUBMED    35673614
REFERENCE   2  (bases 1 to 5984)
  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: "Synth Biol 
            (Oxf)."; date: "2022-05-3"; volume: "7(1):ysac007. doi"; pages: " 
            10.1093/synbio/ysac007. eCollection 2022"
COMMENT     Sequence Label: pTJV1Sc-rpoB
FEATURES             Location/Qualifiers
     source          1..5984
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    258..276
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        307..382
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             403..432
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             448..465
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(521..538)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      691..737
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(876..1667)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(1668..1759)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(2036..2079)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    2082..2103
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     RBS             3385..3393
                     /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)"
     CDS             3399..4184
                     /codon_start=1
                     /gene="bet"
                     /product="single-stranded DNA binding recombinase in the 
                     lambda Red system"
                     /label=Beta
                     /note="mediates pairing of a single-stranded DNA with a 
                     complementary target"
                     /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
                     ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
                     TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
                     FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
                     LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
     terminator      4202..4288
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4431..5381)
                     /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      5429..5651
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 ggtctgctat gtggtgctat ctgacttttt gctgttcagc agttcctgcc ctctgatttt
       61 ccagtctgac cacttcggat tatcccgtga caggtcattc agactggcta atgcacccag
      121 taaggcagcg gtatcatcaa cggggtctga cgctcagtgg aacgaaaact cacgttaagg
      181 gattttggtc atgagattat cagctttcgc taaggatgat ttctggaatt cttccctatc
      241 agtgatagag attgacatcc ctatcagtga tagagatact gagcactgtc caatcgaaac
      301 ccctgagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      361 aaaagtggca ccgagtcggt gctttttttg aagcttgggc ccgaacaaaa actcatctca
      421 gaagaggatc tgaatagcgc cgtcgaccat catcatcatc atcattgagt ttaaacggtc
      481 tccagcttgg ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag
      541 aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac
      601 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc
      661 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
      721 tgggcctttc gttttatctg ttgtttgtcg gtgaactgga tccttactcg agtctagact
      781 gcaggcggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
      841 gtatatatga gtaaacttgg tctgctgaca ggacattatt tgccgactac cttggtgatc
      901 tcgcctttca cgtagtggac aaattcttcc aactgatctg cgcgcgaggc caagcgatct
      961 tcttcttgtc caagataagc ctgtctagct tcaagtatga cgggctgata ctgggccggc
     1021 aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc ggttactgcg
     1081 ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct catcgccagc ccagtcgggc
     1141 ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa atagatcctg ttcaggaacc
     1201 ggatcaaaga gttcctccgc cgctggacct accaaggcaa cgctatgttc tcttgctttt
     1261 gtcagcaaga tagccagatc aatgtcgatc gtggctggct cgaagatacc tgcaagaatg
     1321 tcattgcgct gccattctcc aaattgcagt tcgcgcttag ctggataacg ccacggaatg
     1381 atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga gaatctcgct ctctccaggg
     1441 gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt
     1501 acggtcaccg taaccagcaa atcaatatca ctgtgtggct tcaggccgcc atccactgcg
     1561 gagccgtaca aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat gacgccaact
     1621 acctctgata gttgagtcga tacttcggcg atcaccgctt ccctcatact cttccttttt
     1681 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     1741 atttagaaaa ataaacaaat agctagctca ctcggtcgct accagggtta ttgtctcatg
     1801 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttca gcgcacattt
     1861 ccccgaaaag tgccacctgc ttacgtgccg atcaacgtct cattttcgcc agatatcgac
     1921 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     1981 tttcgtcata ggcgtatcac gaggcccttt cgtcataggc gtatcacgag gcagaatttc
     2041 agataaaaaa aatccttagc tttcgctaag gatgatttct ggaattcgcg gccgcttcta
     2101 gagttgacag ctagctcagt cctaggtatg gtgcttaatt aaatgcgcac ccttagcgag
     2161 aggtttatca ttaaggtcaa cctctggatg ttgtttcggc atcctgcatt gaatctgagt
     2221 tactgtctgt tttcctgcac ccgactcact acggtcgcgt atgtccaatc gaaaccttgg
     2281 aaggtccgaa catcggtctg atcaactctc tgtccgtcag gaaacccgtt ttttctgacg
     2341 taagggtgcg caactttcat gaaatccgct gaatatttga acacttttag attgagaaat
     2401 ctcggcctac ctgtcatgaa caatttgcat gacatgtcta aggcgactcg catatctgtt
     2461 gaaacacttc ggttgttaat ctatacagct gattttcgct ataggatcta cactgtagaa
     2521 aagaaaggcc cagagaagag aatgagaacc atttaccaac cttctcgaga acttaaagcc
     2581 ttacaaggat gggttctacg taacatttta gataaactgt cgtcatctcc tttttctatt
     2641 ggatttgaaa agcaccaatc tattttgaat aatgctaccc cgcatattgg ggcaaacttt
     2701 atactgaata ttgatttgga ggattttttc ccaagtttaa ctgctaacaa agtttttgga
     2761 gtgttccatt ctcttggtta taatcgacta atatcttcag ttttgacaaa aatatgttgt
     2821 tataaaaatc tgctaccaca aggtgctcca tcatcaccta aattagctaa tctaatatgt
     2881 tctaaacttg attatcgtat tcagggttat gcaggtagtc ggggcttgat atatacgaga
     2941 tatgccgatg acctcacctt atctgcacag tctatgaaaa aggttgttaa agcacgtgat
     3001 tttttatttt ctataatccc aagtgaagga ttggttatta actcaaaaaa aacttgtatt
     3061 agtgggcctc gtagtcagag gaaagttaca ggtttagtta tttcacaaga gaaagttggg
     3121 ataggtagag aaaaatataa agaaattaga gcaaagatac atcatatatt ttgcggtaag
     3181 tcttctgaga tagaacacgt taggggatgg ttgtcattta ttttaagtgt ggattcaaaa
     3241 agccatagga gattaataac ttatattagc aaattagaaa aaaaatatgg aaagaaccct
     3301 ttaaataaag cgaagaccta aggatccggt tgattggatc caattgacag ctagctcagt
     3361 cctaggtatc attggatcca attataagga ggtaacgaat gagtactgca ctcgcaacgc
     3421 tggctgggaa gctggctgaa cgtgtcggca tggattctgt cgacccacag gaactgatca
     3481 ccactcttcg ccagacggca tttaaaggtg atgccagcga tgcgcagttc atcgcattac
     3541 tgatcgttgc caaccagtac ggccttaatc cgtggacgaa agaaatttac gcctttcctg
     3601 ataagcagaa tggcatcgtt ccggtggtgg gcgttgatgg ctggtcccgc atcatcaatg
     3661 aaaaccagca gtttgatggc atggactttg agcaggacaa tgaatcctgt acatgccgga
     3721 tttaccgcaa ggaccgtaat catccgatct gcgttaccga atggatggat gaatgccgcc
     3781 gcgaaccatt caaaactcgc gaaggcagag aaatcacggg gccgtggcag tcgcatccca
     3841 aacggatgtt acgtcataaa gccatgattc agtgtgcccg tctggccttc ggatttgctg
     3901 gtatctatga caaggatgaa gccgagcgca ttgtcgaaaa tactgcatac actgcagaac
     3961 gtcagccgga acgcgacatc actccggtta acgatgaaac catgcaggag attaacactc
     4021 tgctgatcgc cctggataaa acatgggatg acgacttatt gccgctctgt tcccagatat
     4081 ttcgccgcga cattcgtgca tcgtcagaac tgacacaggc cgaagcagta aaagctcttg
     4141 gattcctgaa acagaaagcc gcagagcaga aggtggcagc atgaacgcgt gctagaggca
     4201 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     4261 ggtgaacgct ctcctgagta ggacaaatcc gccgccctag acctagggcg ttcggctgcg
     4321 gcgagcggta tcagctcgag tctagactgc aggcgtaacg gtgaacagtt gttctacttt
     4381 tgtttgttag tcttgatgct tcactgatag atacaagagc cataagaacc tcagatcctt
     4441 ccgtatttag ccagtatgtt ctctagtgtg gttcgttgtt tttgcgtgag ccatgagaac
     4501 gaaccattga gatcatactt actttgcatg tcactcaaaa attttgcctc aaaactggtg
     4561 agctgaattt ttgcagttaa agcatcgtgt agtgtttttc ttagtccgtt atgtaggtag
     4621 gaatctgatg taatggttgt tggtattttg tcaccattca tttttatctg gttgttctca
     4681 agttcggtta cgagatccat ttgtctatct agttcaactt ggaaaatcaa cgtatcagtc
     4741 gggcggcctc gcttatcaac caccaatttc atattgctgt aagtgtttaa atctttactt
     4801 attggtttca aaacccattg gttaagcctt ttaaactcat ggtagttatt ttcaagcatt
     4861 aacatgaact taaattcatc aaggctaatc tctatatttg ccttgtgagt tttcttttgt
     4921 gttagttctt ttaataacca ctcataaatc ctcatagagt atttgttttc aaaagactta
     4981 acatgttcca gattatattt tatgaatttt tttaactgga aaagataagg caatatctct
     5041 tcactaaaaa ctaattctaa tttttcgctt gagaacttgg catagtttgt ccactggaaa
     5101 atctcaaagc ctttaaccaa aggattcctg atttccacag ttctcgtcat cagctctctg
     5161 gttgctttag ctaatacacc ataagcattt tccctactga tgttcatcat ctgaacgtat
     5221 tggttataag tgaacgatac cgtccgttct ttccttgtag ggttttcaat cgtggggttg
     5281 agtagtgcca cacagcataa aattagcttg gtttcatgct ccgttaagtc atagcgacta
     5341 atcgctagtt catttgcttt gaaaacaact aattcagaca tacatctcaa ttggtctagg
     5401 tgattttaat cactatacca attgagatgg gctagtcaat gataattact agtccttttc
     5461 ctttgagttg tgggtatctg taaattctgc tagacctttg ctggaaaact tgtaaattct
     5521 gctagaccct ctgtaaattc cgctagacct ttgtgtgttt tttttgttta tattcaagtg
     5581 gttataattt atagaataaa gaaagaataa aaaaagataa aaagaataga tcccagccct
     5641 gtgtataact cactacttta gtcagttccg cagtattaca aaaggatgtc gcaaacgctg
     5701 tttgctcctc tacaaaacag accttaaaac cctaaaggct taagtagcac cctcgcaagc
     5761 tcggttgcgg ccgcaatcgg gcaaatcgct gaatattcct tttgtctccg accatcaggc
     5821 acctgagtcg ctgtcttttt cgtgacattc agttcgctgc gctcacggct ctggcagtga
     5881 atgggggtaa atggcactac aggcgccttt tatggattca tgcaaggaaa ctacccataa
     5941 tacaagaaaa gcccgtcacg ggcttctcag ggcgttttat ggcg
//
