LOCUS       sequence-325058-        6328 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; npt2 for kanamycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6328)
  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 6328)
  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: pTJV1ScKm-rpoB
FEATURES             Location/Qualifiers
     source          1..6328
                     /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(1788..2582)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1899..1918)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2509..2528
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     terminator      complement(2981..3024)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    3027..3048
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     RBS             4330..4338
                     /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             4344..5129
                     /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      5147..5233
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5375..6325)
                     /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 gagcactgtc caatcgaaac
      901 ccctgagttt 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 atccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     1441 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcat gcataaaaac
     1501 tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc
     1561 tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catgggggtg
     1621 ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa
     1681 aacgattccg aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg
     1741 caggttgggc gtcgcttggt cggtcatttc gaaccccaga gtcccgctca gaagaactcg
     1801 tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg
     1861 aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct
     1921 atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg
     1981 ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg
     2041 ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc
     2101 tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg
     2161 atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc
     2221 cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga
     2281 tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg
     2341 agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc
     2401 tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc
     2461 gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag
     2521 ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc
     2581 atgcgaaacg atcctcatcc tgtctcttga tcagatcttg atcccctgcg ccatcagatc
     2641 cttggcggca agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc
     2701 gccccagctg gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc
     2761 catgtaagcc cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag
     2821 atagcccagt agccccgaaa agtgccacct gcttacgtgc cgatcaacgt ctcattttcg
     2881 ccagatatcg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     2941 atcacgaggc cctttcgtca taggcgtatc acgaggcaga atttcagata aaaaaaatcc
     3001 ttagctttcg ctaaggatga tttctggaat tcgcggccgc ttctagagtt tacagctagc
     3061 tcagtcctag gtattatgct taattaaatg cgcaccctta gcgagaggtt tatcattaag
     3121 gtcaacctct ggatgttgtt tcggcatcct gcattgaatc tgagttactg tctgttttcc
     3181 tgcacccgac tcactacggt cgcgtatgtc caatcgaaac cttggaaggt ccgaacatcg
     3241 gtctgatcaa ctctctgtcc gtcaggaaac ccgttttttc tgacgtaagg gtgcgcaact
     3301 ttcatgaaat ccgctgaata tttgaacact tttagattga gaaatctcgg cctacctgtc
     3361 atgaacaatt tgcatgacat gtctaaggcg actcgcatat ctgttgaaac acttcggttg
     3421 ttaatctata cagctgattt tcgctatagg atctacactg tagaaaagaa aggcccagag
     3481 aagagaatga gaaccattta ccaaccttct cgagaactta aagccttaca aggatgggtt
     3541 ctacgtaaca ttttagataa actgtcgtca tctccttttt ctattggatt tgaaaagcac
     3601 caatctattt tgaataatgc taccccgcat attggggcaa actttatact gaatattgat
     3661 ttggaggatt ttttcccaag tttaactgct aacaaagttt ttggagtgtt ccattctctt
     3721 ggttataatc gactaatatc ttcagttttg acaaaaatat gttgttataa aaatctgcta
     3781 ccacaaggtg ctccatcatc acctaaatta gctaatctaa tatgttctaa acttgattat
     3841 cgtattcagg gttatgcagg tagtcggggc ttgatatata cgagatatgc cgatgacctc
     3901 accttatctg cacagtctat gaaaaaggtt gttaaagcac gtgatttttt attttctata
     3961 atcccaagtg aaggattggt tattaactca aaaaaaactt gtattagtgg gcctcgtagt
     4021 cagaggaaag ttacaggttt agttatttca caagagaaag ttgggatagg tagagaaaaa
     4081 tataaagaaa ttagagcaaa gatacatcat atattttgcg gtaagtcttc tgagatagaa
     4141 cacgttaggg gatggttgtc atttatttta agtgtggatt caaaaagcca taggagatta
     4201 ataacttata ttagcaaatt agaaaaaaaa tatggaaaga accctttaaa taaagcgaag
     4261 acctaaggat ccggttgatt ggatccaatt gacagctagc tcagtcctag gtatcattgg
     4321 atccaattat aaggaggtaa cgaatgagta ctgcactcgc aacgctggct gggaagctgg
     4381 ctgaacgtgt cggcatggat tctgtcgacc cacaggaact gatcaccact cttcgccaga
     4441 cggcatttaa aggtgatgcc agcgatgcgc agttcatcgc attactgatc gttgccaacc
     4501 agtacggcct taatccgtgg acgaaagaaa tttacgcctt tcctgataag cagaatggca
     4561 tcgttccggt ggtgggcgtt gatggctggt cccgcatcat caatgaaaac cagcagtttg
     4621 atggcatgga ctttgagcag gacaatgaat cctgtacatg ccggatttac cgcaaggacc
     4681 gtaatcatcc gatctgcgtt accgaatgga tggatgaatg ccgccgcgaa ccattcaaaa
     4741 ctcgcgaagg cagagaaatc acggggccgt ggcagtcgca tcccaaacgg atgttacgtc
     4801 ataaagccat gattcagtgt gcccgtctgg ccttcggatt tgctggtatc tatgacaagg
     4861 atgaagccga gcgcattgtc gaaaatactg catacactgc agaacgtcag ccggaacgcg
     4921 acatcactcc ggttaacgat gaaaccatgc aggagattaa cactctgctg atcgccctgg
     4981 ataaaacatg ggatgacgac ttattgccgc tctgttccca gatatttcgc cgcgacattc
     5041 gtgcatcgtc agaactgaca caggccgaag cagtaaaagc tcttggattc ctgaaacaga
     5101 aagccgcaga gcagaaggtg gcagcatgaa cgcgtgctag aggcatcaaa taaaacgaaa
     5161 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     5221 gagtaggaca aatccgccgc cctagaccta ggcgttcggc tgcggcgagc ggtatcagct
     5281 cgagtctaga ctgcaggcgt aacggtgaac agttgttcta cttttgtttg ttagtcttga
     5341 tgcttcactg atagatacaa gagccataag aacctcagat ccttccgtat ttagccagta
     5401 tgttctctag tgtggttcgt tgtttttgcg tgagccatga gaacgaacca ttgagatcat
     5461 acttactttg catgtcactc aaaaattttg cctcaaaact ggtgagctga atttttgcag
     5521 ttaaagcatc gtgtagtgtt tttcttagtc cgttacgtag gtaggaatct gatgtaatgg
     5581 ttgttggtat tttgtcacca ttcattttta tctggttgtt ctcaagttcg gttacgagat
     5641 ccatttgtct atctagttca acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat
     5701 caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc
     5761 attggttaag ccttttaaac tcatggtagt tattttcaag cattaacatg aacttaaatt
     5821 catcaaggct aatctctata tttgccttgt gagttttctt ttgtgttagt tcttttaata
     5881 accactcata aatcctcata gagtatttgt tttcaaaaga cttaacatgt tccagattat
     5941 attttatgaa tttttttaac tggaaaagat aaggcaatat ctcttcacta aaaactaatt
     6001 ctaatttttc gcttgagaac ttggcatagt ttgtccactg gaaaatctca aagcctttaa
     6061 ccaaaggatt cctgatttcc acagttctcg tcatcagctc tctggttgct ttagctaata
     6121 caccataagc attttcccta ctgatgttca tcatctgagc gtattggtta taagtgaacg
     6181 ataccgtccg ttctttcctt gtagggtttt caatcgtggg gttgagtagt gccacacagc
     6241 ataaaattag cttggtttca tgctccgtta agtcatagcg actaatcgct agttcatttg
     6301 ctttgaaaac aactaattca gacataca
//
