LOCUS       sequence-331330-        5899 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; aacC1 for gentamycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5899)
  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 5899)
  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: pTJV1ScGm-rpoB
FEATURES             Location/Qualifiers
     source          1..5899
                     /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"
     promoter        join(843..848,849..865,866..871)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             1060..1593
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     terminator      complement(1951..1994)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    1997..2018
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     RBS             3300..3308
                     /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             3314..4099
                     /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      4117..4203
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4346..5296)
                     /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      5344..5566
                     /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 agtctagccg
      781 ccttgcgtat aatatttgcc catggacgca caccgtggaa acggatgaag gcacgaaccc
      841 agttgacata agcctgttcg gttcgtaaac tgtaatgcaa gtagcgtatg cgctcacgca
      901 actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg
      961 cttgttatga ctgttttttt gtacagtcta tgcctcgggc atccaagcag caagcgcgtt
     1021 acgccgtggg tcgatgtttg atgttatgga gcagcaacga tgttacgcag cagcaacgat
     1081 gttacgcagc agggcagtcg ccctaaaaca aagttaggtg gctcaagtat gggcatcatt
     1141 cgcacatgta ggctcggccc tgaccaagtc aaatccatgc gggctgctct tgatcttttc
     1201 ggtcgtgagt tcggagacgt agccacctac tcccaacatc agccggactc cgattacctc
     1261 gggaacttgc tccgtagtaa gacattcatc gcgcttgctg ccttcgacca agaagcggtt
     1321 gttggcgctc tcgcggctta cgttctgccc aagtttgagc agccgcgtag tgagatctat
     1381 atctatgatc tcgcagtctc cggagagcac cggaggcagg gcattgccac cgcgctcatc
     1441 aatctcctca agcatgaggc caacgcgctt ggtgcttatg tgatctacgt gcaagcagat
     1501 tacggtgacg atcccgcagt ggctctctat acaaagttgg gcatacggga agaagtgatg
     1561 cactttgata tcgacccaag taccgccacc taacaattcg ttcaagccga gatcggcttc
     1621 ccggccgcgg agttgttcgg taaattgtca caacgccgca ggtggcactt ttcgggtcta
     1681 gctcactcgg tcgctaccag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     1741 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgcttacg
     1801 tgccgatcaa cgtctcattt tcgccagata tcgacgtcta agaaaccatt attatcatga
     1861 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcataggcgt atcacgaggc
     1921 cctttcgtca taggcgtatc acgaggcaga atttcagata aaaaaaatcc ttagctttcg
     1981 ctaaggatga tttctggaat tcgcggccgc ttctagagtt gacagctagc tcagtcctag
     2041 gtatggtgct taattaaatg cgcaccctta gcgagaggtt tatcattaag gtcaacctct
     2101 ggatgttgtt tcggcatcct gcattgaatc tgagttactg tctgttttcc tgcacccgac
     2161 tcactacggt cgcgtatgtc caatcgaaac cttggaaggt ccgaacatcg gtctgatcaa
     2221 ctctctgtcc gtcaggaaac ccgttttttc tgacgtaagg gtgcgcaact ttcatgaaat
     2281 ccgctgaata tttgaacact tttagattga gaaatctcgg cctacctgtc atgaacaatt
     2341 tgcatgacat gtctaaggcg actcgcatat ctgttgaaac acttcggttg ttaatctata
     2401 cagctgattt tcgctatagg atctacactg tagaaaagaa aggcccagag aagagaatga
     2461 gaaccattta ccaaccttct cgagaactta aagccttaca aggatgggtt ctacgtaaca
     2521 ttttagataa actgtcgtca tctccttttt ctattggatt tgaaaagcac caatctattt
     2581 tgaataatgc taccccgcat attggggcaa actttatact gaatattgat ttggaggatt
     2641 ttttcccaag tttaactgct aacaaagttt ttggagtgtt ccattctctt ggttataatc
     2701 gactaatatc ttcagttttg acaaaaatat gttgttataa aaatctgcta ccacaaggtg
     2761 ctccatcatc acctaaatta gctaatctaa tatgttctaa acttgattat cgtattcagg
     2821 gttatgcagg tagtcggggc ttgatatata cgagatatgc cgatgacctc accttatctg
     2881 cacagtctat gaaaaaggtt gttaaagcac gtgatttttt attttctata atcccaagtg
     2941 aaggattggt tattaactca aaaaaaactt gtattagtgg gcctcgtagt cagaggaaag
     3001 ttacaggttt agttatttca caagagaaag ttgggatagg tagagaaaaa tataaagaaa
     3061 ttagagcaaa gatacatcat atattttgcg gtaagtcttc tgagatagaa cacgttaggg
     3121 gatggttgtc atttatttta agtgtggatt caaaaagcca taggagatta ataacttata
     3181 ttagcaaatt agaaaaaaaa tatggaaaga accctttaaa taaagcgaag acctaaggat
     3241 ccggttgatt ggatccaatt gacagctagc tcagtcctag gtatcattgg atccaattat
     3301 aaggaggtaa cgaatgagta ctgcactcgc aacgctggct gggaagctgg ctgaacgtgt
     3361 cggcatggat tctgtcgacc cacaggaact gatcaccact cttcgccaga cggcatttaa
     3421 aggtgatgcc agcgatgcgc agttcatcgc attactgatc gttgccaacc agtacggcct
     3481 taatccgtgg acgaaagaaa tttacgcctt tcctgataag cagaatggca tcgttccggt
     3541 ggtgggcgtt gatggctggt cccgcatcat caatgaaaac cagcagtttg atggcatgga
     3601 ctttgagcag gacaatgaat cctgtacatg ccggatttac cgcaaggacc gtaatcatcc
     3661 gatctgcgtt accgaatgga tggatgaatg ccgccgcgaa ccattcaaaa ctcgcgaagg
     3721 cagagaaatc acggggccgt ggcagtcgca tcccaaacgg atgttacgtc ataaagccat
     3781 gattcagtgt gcccgtctgg ccttcggatt tgctggtatc tatgacaagg atgaagccga
     3841 gcgcattgtc gaaaatactg catacactgc agaacgtcag ccggaacgcg acatcactcc
     3901 ggttaacgat gaaaccatgc aggagattaa cactctgctg atcgccctgg ataaaacatg
     3961 ggatgacgac ttattgccgc tctgttccca gatatttcgc cgcgacattc gtgcatcgtc
     4021 agaactgaca caggccgaag cagtaaaagc tcttggattc ctgaaacaga aagccgcaga
     4081 gcagaaggtg gcagcatgaa cgcgtgctag aggcatcaaa taaaacgaaa ggctcagtcg
     4141 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     4201 aatccgccgc cctagaccta gggcgttcgg ctgcggcgag cggtatcagc tcgagtctag
     4261 actgcaggcg taacggtgaa cagttgttct acttttgttt gttagtcttg atgcttcact
     4321 gatagataca agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta
     4381 gtgtggttcg ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt
     4441 gcatgtcact caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat
     4501 cgtgtagtgt ttttcttagt ccgttatgta ggtaggaatc tgatgtaatg gttgttggta
     4561 ttttgtcacc attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc
     4621 tatctagttc aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca
     4681 atttcatatt gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa
     4741 gccttttaaa ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc
     4801 taatctctat atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat
     4861 aaatcctcat agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga
     4921 atttttttaa ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt
     4981 cgcttgagaa cttggcatag tttgtccact ggaaaatctc aaagccttta accaaaggat
     5041 tcctgatttc cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag
     5101 cattttccct actgatgttc atcatctgaa cgtattggtt ataagtgaac gataccgtcc
     5161 gttctttcct tgtagggttt tcaatcgtgg ggttgagtag tgccacacag cataaaatta
     5221 gcttggtttc atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa
     5281 caactaattc agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga
     5341 gatgggctag tcaatgataa ttactagtcc ttttcctttg agttgtgggt atctgtaaat
     5401 tctgctagac ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta
     5461 gacctttgtg tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag
     5521 aataaaaaaa gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag
     5581 ttccgcagta ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt
     5641 aaaaccctaa aggcttaagt agcaccctcg caagctcggt tgcggccgca atcgggcaaa
     5701 tcgctgaata ttccttttgt ctccgaccat caggcacctg agtcgctgtc tttttcgtga
     5761 cattcagttc gctgcgctca cggctctggc agtgaatggg ggtaaatggc actacaggcg
     5821 ccttttatgg attcatgcaa ggaaactacc cataatacaa gaaaagcccg tcacgggctt
     5881 ctcagggcgt tttatggcg
//
