LOCUS       sequence-325037-        5920 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Generates ssDNA in vivo targeting tetA for recombineering w/ CspRecT
            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 5920)
  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 5920)
  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: pTJV1Sc2-tetA
FEATURES             Location/Qualifiers
     source          1..5920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      45..267
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     protein_bind    833..851
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    858..876
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        907..982
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1003..1032
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1048..1065
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1121..1138)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1291..1337
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1473..2264)
                     /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(2286..2390)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(2546..2589)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    2592..2613
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     RBS             3895..3903
                     /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      4739..4825
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4967..5917)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
ORIGIN
        1 tctcaattgg tctaggtgat tttaatcact ataccaattg agatgggcta gtcaatgata
       61 attactagtc cttttccttt gagttgtggg tatctgtaaa ttctgctaga cctttgctgg
      121 aaaacttgta aattctgcta gaccctctgt aaattccgct agacctttgt gtgttttttt
      181 tgtttatatt caagtggtta taatttatag aataaagaaa gaataaaaaa agataaaaag
      241 aatagatccc agccctgtgt ataactcact actttagtca gttccgcagt attacaaaag
      301 gatgtcgcaa acgctgtttg ctcctctaca aaacagacct taaaacccta aaggcttaag
      361 tagcaccctc gcaagctcgg ttgcggccgc aatcgggcaa atcgctgaat attccttttg
      421 tctccgacca tcaggcacct gagtcgctgt ctttttcgtg acattcagtt cgctgcgctc
      481 acggctctgg cagtgaatgg gggtaaatgg cactacaggc gccttttatg gattcatgca
      541 aggaaactac ccataataca agaaaagccc gtcacgggct tctcagggcg ttttatggcg
      601 ggtctgctat gtggtgctat ctgacttttt gctgttcagc agttcctgcc ctctgatttt
      661 ccagtctgac cacttcggat tatcccgtga caggtcattc agactggcta atgcacccag
      721 taaggcagcg gtatcatcaa cggggtctga cgctcagtgg aacgaaaact cacgttaagg
      781 gattttggtc atgagattat cagctttcgc taaggatgat ttctggaatt cttccctatc
      841 agtgatagag attgacatcc ctatcagtga tagagatact gagcaccgcc agcagaatgc
      901 cttagtgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      961 aaaagtggca ccgagtcggt gctttttttg aagcttgggc ccgaacaaaa actcatctca
     1021 gaagaggatc tgaatagcgc cgtcgaccat catcatcatc atcattgagt ttaaacggtc
     1081 tccagcttgg ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag
     1141 aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac
     1201 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc
     1261 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
     1321 tgggcctttc gttttatctg ttgtttgtcg gtgaactgga tccttactcg agtctagact
     1381 gcaggcggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     1441 gtatatatga gtaaacttgg tctgacagga cattatttgc cgactacctt ggtgatctcg
     1501 cctttcacgt agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct
     1561 tcttgtccaa gataagcctg tctagcttca agtatgacgg gctgatactg ggccggcagg
     1621 cgctccattg cccagtcggc agcgacatcc ttcggcgcga ttttgccggt tactgcgctg
     1681 taccaaatgc gggacaacgt aagcactaca tttcgctcat cgccagccca gtcgggcggc
     1741 gagttccata gcgttaaggt ttcatttagc gcctcaaata gatcctgttc aggaaccgga
     1801 tcaaagagtt cctccgccgc tggacctacc aaggcaacgc tatgttctct tgcttttgtc
     1861 agcaagatag ccagatcaat gtcgatcgtg gctggctcga agatacctgc aagaatgtca
     1921 ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg gataacgcca cggaatgatg
     1981 tcgtcgtgca caacaatggt gacttctaca gcgcggagaa tctcgctctc tccaggggaa
     2041 gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg
     2101 gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca ggccgccatc cactgcggag
     2161 ccgtacaaat gtacggccag caacgtcggt tcgagatggc gctcgatgac gccaactacc
     2221 tctgatagtt gagtcgatac ttcggcgatc accgcttccc tcatgctagc tcactcggtc
     2281 gctacactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     2341 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     2401 cgaaaagtgc cacctgctta cgtgccgatc aacgtctcat tttcgccaga tatcgacgtc
     2461 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     2521 cgtcataggc gtatcacgag gcagaatttc agataaaaaa aatccttagc tttcgctaag
     2581 gatgatttct ggaattcgcg gccgcttcta gagttgacag ctagctcagt cctaggtatg
     2641 gtgcttaatt aaatgcgcac ccttagcgag aggtttatca ttaaggtcaa cctctggatg
     2701 ttgtttcggc atcctgcatt gaatctgagt tactgtctgt tttcctgcct gcgcgatctg
     2761 gttcactcga acgacgtcac cgcccactat ggcattctgc tggcgctgta tgcgttggtg
     2821 caatttgcag gaaacccgtt ttttctgacg taagggtgcg caactttcat gaaatccgct
     2881 gaatatttga acacttttag attgagaaat ctcggcctac ctgtcatgaa caatttgcat
     2941 gacatgtcta aggcgactcg catatctgtt gaaacacttc ggttgttaat ctatacagct
     3001 gattttcgct ataggatcta cactgtagaa aagaaaggcc cagagaagag aatgagaacc
     3061 atttaccaac cttctcgaga acttaaagcc ttacaaggat gggttctacg taacatttta
     3121 gataaactgt cgtcatctcc tttttctatt ggatttgaaa agcaccaatc tattttgaat
     3181 aatgctaccc cgcatattgg ggcaaacttt atactgaata ttgatttgga ggattttttc
     3241 ccaagtttaa ctgctaacaa agtttttgga gtgttccatt ctcttggtta taatcgacta
     3301 atatcttcag ttttgacaaa aatatgttgt tataaaaatc tgctaccaca aggtgctcca
     3361 tcatcaccta aattagctaa tctaatatgt tctaaacttg attatcgtat tcagggttat
     3421 gcaggtagtc ggggcttgat atatacgaga tatgccgatg acctcacctt atctgcacag
     3481 tctatgaaaa aggttgttaa agcacgtgat tttttatttt ctataatccc aagtgaagga
     3541 ttggttatta actcaaaaaa aacttgtatt agtgggcctc gtagtcagag gaaagttaca
     3601 ggtttagtta tttcacaaga gaaagttggg ataggtagag aaaaatataa agaaattaga
     3661 gcaaagatac atcatatatt ttgcggtaag tcttctgaga tagaacacgt taggggatgg
     3721 ttgtcattta ttttaagtgt ggattcaaaa agccatagga gattaataac ttatattagc
     3781 aaattagaaa aaaaatatgg aaagaaccct ttaaataaag cgaagaccta aggatccggt
     3841 tgattggatc caattgacag ctagctcagt cctaggtacc attggatcca attataagga
     3901 ggtaacgaat gaaccaaatc gtgaagttca ctgacgactc tggcctggcg gttcaagtta
     3961 ctccagacga tgttcgccgt tatatctgtg agaacgctac tgaaaaagag gtgggcctct
     4021 ttctgcaact ctgtcagact caacgtctga atccgtttgt gaaagacgct tacctggtga
     4081 aatacggcgg tgctccagct tctatgatta cttcctatca agtttttaac cgtcgcgcgt
     4141 gtcgtgatgc taactatgat ggtatcaaat ctggtgtggt tgttctgcgt gacggtgatg
     4201 ttgtgcataa acgtggtgct gcgtgctaca aaaaggcggg tgaggagctc atcggtggtt
     4261 gggcggaagt tcgctttaag gatggccgcg agactgcgta tgctgaggtg gcgctcgacg
     4321 actattccac cggcaaatct aattgggcga aaatgccggg tgttatgatc gaaaaatgcg
     4381 cgaaggctgc tgcttggcgc ctcgcgttcc cggacacttt tcagggcatg tacgctgcgg
     4441 aggaaatgga tcaagcgcaa cagccagaac aggtgcgcgc tcaggcggag caaccagtgg
     4501 atctccagcc aatccgcgaa ctcttcaagc catattgcga acacttcggc atcactccgg
     4561 ctgagggtat gactgctgtt tgtggtgcgg tgggcgctga aggcatgcac tctatgaccg
     4621 agcagcaagc tcgccgtgct cgcgcttgga tggaggaaga aatggctgcg ccagctgtgg
     4681 aagcggagta tgaggttgtt gacgagggcg aggtgtttta aacgcgtgct agaggcatca
     4741 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     4801 gaacgctctc ctgagtagga caaatccgcc gccctagacc taggcgttcg gctgcggcga
     4861 gcggtatcag ctcgagtcta gactgcaggc gtaacggtga acagttgttc tacttttgtt
     4921 tgttagtctt gatgcttcac tgatagatac aagagccata agaacctcag atccttccgt
     4981 atttagccag tatgttctct agtgtggttc gttgtttttg cgtgagccat gagaacgaac
     5041 cattgagatc atacttactt tgcatgtcac tcaaaaattt tgcctcaaaa ctggtgagct
     5101 gaatttttgc agttaaagca tcgtgtagtg tttttcttag tccgttacgt aggtaggaat
     5161 ctgatgtaat ggttgttggt attttgtcac cattcatttt tatctggttg ttctcaagtt
     5221 cggttacgag atccatttgt ctatctagtt caacttggaa aatcaacgta tcagtcgggc
     5281 ggcctcgctt atcaaccacc aatttcatat tgctgtaagt gtttaaatct ttacttattg
     5341 gtttcaaaac ccattggtta agccttttaa actcatggta gttattttca agcattaaca
     5401 tgaacttaaa ttcatcaagg ctaatctcta tatttgcctt gtgagttttc ttttgtgtta
     5461 gttcttttaa taaccactca taaatcctca tagagtattt gttttcaaaa gacttaacat
     5521 gttccagatt atattttatg aattttttta actggaaaag ataaggcaat atctcttcac
     5581 taaaaactaa ttctaatttt tcgcttgaga acttggcata gtttgtccac tggaaaatct
     5641 caaagccttt aaccaaagga ttcctgattt ccacagttct cgtcatcagc tctctggttg
     5701 ctttagctaa tacaccataa gcattttccc tactgatgtt catcatctga gcgtattggt
     5761 tataagtgaa cgataccgtc cgttctttcc ttgtagggtt ttcaatcgtg gggttgagta
     5821 gtgccacaca gcataaaatt agcttggttt catgctccgt taagtcatag cgactaatcg
     5881 ctagttcatt tgctttgaaa acaactaatt cagacataca
//
