LOCUS       sequence-331333-        6276 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 6276)
  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 6276)
  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..6276
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    342..363
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     terminator      366..409
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             965..1759
                     /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(1019..1038)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1629..1648
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      complement(2000..2046)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2199..2216
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(2272..2289)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2305..2334)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_RNA        complement(2355..2430)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    2461..2479
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2486..2504
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     rep_origin      3070..3292
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             3340..4290
                     /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"
     terminator      complement(4433..4519)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4537..5322)
                     /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"
     RBS             5328..5336
                     /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)"
ORIGIN
        1 gccttagaca tgtcatgcaa attgttcatg acaggtaggc cgagatttct caatctaaaa
       61 gtgttcaaat attcagcgga tttcatgaaa gttgcgcacc cttacgtcag aaaaaacggg
      121 tttcctgacg gacagagagt tgatcagacc gatgttcgga ccttccaagg tttcgattgg
      181 acatacgcga ccgtagtgag tcgggtgcag gaaaacagac agtaactcag attcaatgca
      241 ggatgccgaa acaacatcca gaggttgacc ttaatgataa acctctcgct aagggtgcgc
      301 atttaattaa gcaccatacc taggactgag ctagctgtca actctagaag cggccgcgaa
      361 ttccagaaat catccttagc gaaagctaag gatttttttt atctgaaatt ctgcctcgtg
      421 atacgcctat gacgaaaggg cctcgtgata cgcctatgac gaaagggcct cgtgatacgc
      481 ctatttttat aggttaatgt catgataata atggtttctt agacgtcgat atctggcgaa
      541 aatgagacgt tgatcggcac gtaagcaggt ggcacttttc ggggaaatgt gcgcggaacc
      601 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
      661 tggtagcgac cgagtgagct agacccgaaa agtgcaacct gggatgaatg tcagctactg
      721 ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct
      781 tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc
      841 tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc
      901 gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat gaggatcgtt
      961 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     1021 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     1081 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     1141 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     1201 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     1261 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     1321 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     1381 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     1441 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     1501 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     1561 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     1621 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     1681 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     1741 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     1801 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     1861 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     1921 ttcgcccacc cccatgggca aatattatac gcaaggcggc tagactcgag taaggatcca
     1981 gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt
     2041 tatttgatgc ctggcagttc cctactctcg catggggaga ccccacacta ccatcggcgc
     2101 tacggcgttt cacttctgag ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc
     2161 caggcaaatt ctgttttatc agaccgcttc tgcgttctga tttaatctgt atcaggctga
     2221 aaatcttctc tcatccgcca aaacagccaa gctggagacc gtttaaactc aatgatgatg
     2281 atgatgatgg tcgacggcgc tattcagatc ctcttctgag atgagttttt gttcgggccc
     2341 aagcttcaaa aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt
     2401 attttaactt gctatttcta gctctaaaac tcaggggttt cgattggaca gtgctcagta
     2461 tctctatcac tgatagggat gtcaatctct atcactgata gggaagaatt ccagaaatca
     2521 tccttagcga aagctgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2581 tgagcgtcag accccgttga tgataccgct gccttactgg gtgcattagc cagtctgaat
     2641 gacctgtcac gggataatcc gaagtggtca gactggaaaa tcagagggca ggaactgctg
     2701 aacagcaaaa agtcagatag caccacatag cagacccgcc ataaaacgcc ctgagaagcc
     2761 cgtgacgggc ttttcttgta ttatgggtag tttccttgca tgaatccata aaaggcgcct
     2821 gtagtgccat ttacccccat tcactgccag agccgtgagc gcagcgaact gaatgtcacg
     2881 aaaaagacag cgactcaggt gcctgatggt cggagacaaa aggaatattc agcgatttgc
     2941 ccgattgcgg ccgcaaccga gcttgcgagg gtgctactta agcctttagg gttttaaggt
     3001 ctgttttgta gaggagcaaa cagcgtttgc gacatccttt tgtaatactg cggaactgac
     3061 taaagtagtg agttatacac agggctggga tctattcttt ttatcttttt ttattctttc
     3121 tttattctat aaattataac cacttgaata taaacaaaaa aaacacacaa aggtctagcg
     3181 gaatttacag agggtctagc agaatttaca agttttccag caaaggtcta gcagaattta
     3241 cagataccca caactcaaag gaaaaggact agtaattatc attgactagc ccatctcaat
     3301 tggtatagtg attaaaatca cctagaccaa ttgagatgta tgtctgaatt agttgttttc
     3361 aaagcaaatg aactagcgat tagtcgctat gacttaacgg agcatgaaac caagctaatt
     3421 ttatgctgtg tggcactact caaccccacg attgaaaacc ctacaaggaa agaacggacg
     3481 gtatcgttca cttataacca atacgttcag atgatgaaca tcagtaggga aaatgcttat
     3541 ggtgtattag ctaaagcaac cagagagctg atgacgagaa ctgtggaaat caggaatcct
     3601 ttggttaaag gctttgagat tttccagtgg acaaactatg ccaagttctc aagcgaaaaa
     3661 ttagaattag tttttagtga agagatattg ccttatcttt tccagttaaa aaaattcata
     3721 aaatataatc tggaacatgt taagtctttt gaaaacaaat actctatgag gatttatgag
     3781 tggttattaa aagaactaac acaaaagaaa actcacaagg caaatataga gattagcctt
     3841 gatgaattta agttcatgtt aatgcttgaa aataactacc atgagtttaa aaggcttaac
     3901 caatgggttt tgaaaccaat aagtaaagat ttaaacactt acagcaatat gaaattggtg
     3961 gttgataagc gaggccgccc gactgatacg ttgattttcc aagttgaact agatagacaa
     4021 atggatctcg taaccgaact tgagaacaac cagataaaaa tgaatggtga caaaatacca
     4081 acaaccatta catcagattc ctacctacat aacggactaa gaaaaacact acacgatgct
     4141 ttaactgcaa aaattcagct caccagtttt gaggcaaaat ttttgagtga catgcaaagt
     4201 aagtatgatc tcaatggttc gttctcatgg ctcacgcaaa aacaacgaac cacactagag
     4261 aacatactgg ctaaatacgg aaggatctga ggttcttatg gctcttgtat ctatcagtga
     4321 agcatcaaga ctaacaaaca aaagtagaac aactgttcac cgttacgcct gcagtctaga
     4381 ctcgagctga taccgctcgc cgcagccgaa cgccctaggt ctagggcggc ggatttgtcc
     4441 tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact
     4501 gagcctttcg ttttatttga tgcctctagc acgcgttcat gctgccacct tctgctctgc
     4561 ggctttctgt ttcaggaatc caagagcttt tactgcttcg gcctgtgtca gttctgacga
     4621 tgcacgaatg tcgcggcgaa atatctggga acagagcggc aataagtcgt catcccatgt
     4681 tttatccagg gcgatcagca gagtgttaat ctcctgcatg gtttcatcgt taaccggagt
     4741 gatgtcgcgt tccggctgac gttctgcagt gtatgcagta ttttcgacaa tgcgctcggc
     4801 ttcatccttg tcatagatac cagcaaatcc gaaggccaga cgggcacact gaatcatggc
     4861 tttatgacgt aacatccgtt tgggatgcga ctgccacggc cccgtgattt ctctgccttc
     4921 gcgagttttg aatggttcgc ggcggcattc atccatccat tcggtaacgc agatcggatg
     4981 attacggtcc ttgcggtaaa tccggcatgt acaggattca ttgtcctgct caaagtccat
     5041 gccatcaaac tgctggtttt cattgatgat gcgggaccag ccatcaacgc ccaccaccgg
     5101 aacgatgcca ttctgcttat caggaaaggc gtaaatttct ttcgtccacg gattaaggcc
     5161 gtactggttg gcaacgatca gtaatgcgat gaactgcgca tcgctggcat cacctttaaa
     5221 tgccgtctgg cgaagagtgg tgatcagttc ctgtgggtcg acagaatcca tgccgacacg
     5281 ttcagccagc ttcccagcca gcgttgcgag tgcagtactc attcgttacc tccttataat
     5341 tggatccaat gatacctagg actgagctag ctgtcaattg gatccaatca accggatcct
     5401 taggtcttcg ctttatttaa agggttcttt ccatattttt tttctaattt gctaatataa
     5461 gttattaatc tcctatggct ttttgaatcc acacttaaaa taaatgacaa ccatccccta
     5521 acgtgttcta tctcagaaga cttaccgcaa aatatatgat gtatctttgc tctaatttct
     5581 ttatattttt ctctacctat cccaactttc tcttgtgaaa taactaaacc tgtaactttc
     5641 ctctgactac gaggcccact aatacaagtt ttttttgagt taataaccaa tccttcactt
     5701 gggattatag aaaataaaaa atcacgtgct ttaacaacct ttttcataga ctgtgcagat
     5761 aaggtgaggt catcggcata tctcgtatat atcaagcccc gactacctgc ataaccctga
     5821 atacgataat caagtttaga acatattaga ttagctaatt taggtgatga tggagcacct
     5881 tgtggtagca gatttttata acaacatatt tttgtcaaaa ctgaagatat tagtcgatta
     5941 taaccaagag aatggaacac tccaaaaact ttgttagcag ttaaacttgg gaaaaaatcc
     6001 tccaaatcaa tattcagtat aaagtttgcc ccaatatgcg gggtagcatt attcaaaata
     6061 gattggtgct tttcaaatcc aatagaaaaa ggagatgacg acagtttatc taaaatgtta
     6121 cgtagaaccc atccttgtaa ggctttaagt tctcgagaag gttggtaaat ggttctcatt
     6181 ctcttctctg ggcctttctt ttctacagtg tagatcctat agcgaaaatc agctgtatag
     6241 attaacaacc gaagtgtttc aacagatatg cgagtc
//
