LOCUS       sequence-483599-        7938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RSF1010 origin of replication plasmid containing Eco1 recombitron 
            with a SapI flanked stuffer in the ncRNA expressed by J23115 
            constitutive promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7938)
  AUTHORS   Gonzalez-Delgado A, Bonillo-Lopez L, Johnson MS, Knodlseder N, Ko 
            CC, Lekbach Y, Oh JH, Selvakumar H, Wold MC, Yu Z, Aragon V, 
            Gralnick JA, Guell M, Hatfull GF, Keitz BK, Koskella B, Mutalik VK, 
            van Pijkeren JP, Shipman SL
  TITLE     Genome editing of phylogenetically distinct bacteria using portable 
            retron-mediated recombineering
  JOURNAL   bioRxiv 2025.06.16.660010
REFERENCE   2  (bases 1 to 7938)
  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: "bioRxiv 
            2025.06.16.660010"
COMMENT     Sequence Label: pREC1010C-Eco1
FEATURES             Location/Qualifiers
     source          1..7938
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             104..943
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein A of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepA
                     /translation="MATHKPINILEAFAAAPPPLDYVLPNMVAGTVGALVSPGGAGKSM
                     LALQLAAQIAGGPDLLEVGELPTGPVIYLPAEDPPTAIHHRLHALGAHLSAEERQAVAD
                     GLLIQPLIGSLPNIMAPEWFDGLKRAAEGRRLMVLDTLRRFHIEEENASGPMAQVIGRM
                     EAIAADTGCSIVFLHHASKGAAMMGAGDQQQASRGSSVLVDNIRWQSYLSSMTSAEAEE
                     WGVDDDQRRFFVRFGVSKANYGAPFADRWFRRHDGGVLKPAVLERQRKSKGVPRGEA"
     CDS             930..1781
                     /codon_start=1
                     /gene="repC"
                     /product="replication protein C of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepC
                     /translation="MVKPKNKHSLSHVRHDPAHCLAPGLFRALKRGERKRSKLDVTYDY
                     GDGKRIEFSGPEPLGADDLRILQGLVAMAGPNGLVLGPEPKTEGGRQLRLFLEPKWEAV
                     TADAMVVKGSYRALAKEIGAEVDSGGALKHIQDCIERLWKVSIIAQNGRKRQGFRLLSE
                     YASDEADGRLYVALNPLIAQAVMGGGQHVRISMDEVRALDSETARLLHQRLCGWIDPGK
                     TGKASIDTLCGYVWPSEASGSTMRKRRQRVREALPELVALGWTVTEFAAGKYDITRPKA
                     AG"
     oriT            1792..1900
                     /note="incP origin of transfer"
     CDS             complement(2067..2882)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2874..2893
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      complement(3014..3108)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     RBS             4554..4562
                     /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)"
     RBS             5794..5816
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        complement(5825..5859)
                     /label=BBa_J23115
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.15"
     terminator      5891..5977
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      6059..6453
                     /label=RSF1010 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     RSF1010; requires the RSF1010 RepA/B/C proteins for 
                     replication (Scholz et al., 1989)"
     CDS             6560..7531
                     /codon_start=1
                     /gene="repB"
                     /product="replication protein B of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepB
                     /translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDATTGQMMNREWSAAEVL
                     QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
                     YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
                     EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
ORIGIN
        1 acgaggcgct gcgcgagtgc ctggaagaac tcagagcggc gcagggcggg ggtagtgacc
       61 ccgccagcgc ctaaccacca actgcctgca aaggaggcaa tcaatggcta cccataagcc
      121 tatcaatatt ctggaggcgt tcgcagcagc gccgccaccg ctggactacg ttttgcccaa
      181 catggtggcc ggtacggtcg gggcgctggt gtcgcccggt ggtgccggta aatccatgct
      241 ggccctgcaa ctggccgcac agattgcagg cgggccggat ctgctggagg tgggcgaact
      301 gcccaccggc ccggtgatct acctgcccgc cgaagacccg cccaccgcca ttcatcaccg
      361 cctgcacgcc cttggggcgc acctcagcgc cgaggaacgg caagccgtgg ctgacggcct
      421 gctgatccag ccgctgatcg gcagcctgcc caacatcatg gccccggagt ggttcgacgg
      481 cctcaagcgc gccgccgagg gccgccgcct gatggtgctg gacacgctgc gccggttcca
      541 catcgaggaa gaaaacgcca gcggccccat ggcccaggtc atcggtcgca tggaggccat
      601 cgccgccgat accgggtgct ctatcgtgtt cctgcaccat gccagcaagg gcgcggccat
      661 gatgggcgca ggcgaccagc agcaggccag ccggggcagc tcggtactgg tcgataacat
      721 ccgctggcag tcctacctgt cgagcatgac cagcgccgag gccgaggaat ggggtgtgga
      781 cgacgaccag cgccggttct tcgtccgctt cggtgtgagc aaggccaact atggcgcacc
      841 gttcgctgat cggtggttca ggcggcatga cggcggggtg ctcaagcccg ccgtgctgga
      901 gaggcagcgc aagagcaagg gggtgccccg tggtgaagcc taagaacaag cacagcctca
      961 gccacgtccg gcacgacccg gcgcactgtc tggcccccgg cctgttccgt gccctcaagc
     1021 ggggcgagcg caagcgcagc aagctggacg tgacgtatga ctacggcgac ggcaagcgga
     1081 tcgagttcag cggcccggag ccgctgggcg ctgatgatct gcgcatcctg caagggctgg
     1141 tggccatggc tgggcctaat ggcctagtgc ttggcccgga acccaagacc gaaggcggac
     1201 ggcagctccg gctgttcctg gaacccaagt gggaggccgt caccgctgat gccatggtgg
     1261 tcaaaggtag ctatcgggcg ctggcaaagg aaatcggggc agaggtcgat agtggtgggg
     1321 cgctcaagca catacaggac tgcatcgagc gcctttggaa ggtatccatc atcgcccaga
     1381 atggccgcaa gcggcagggg tttcggctgc tgtcggagta cgccagcgac gaggcggacg
     1441 ggcgcctgta cgtggccctg aaccccttga tcgcgcaggc cgtcatgggt ggcggccagc
     1501 atgtgcgcat cagcatggac gaggtgcggg cgctggacag cgaaaccgcc cgcctgctgc
     1561 accagcggct gtgtggctgg atcgaccccg gcaaaaccgg caaggcttcc atagatacct
     1621 tgtgcggcta tgtctggccg tcagaggcca gtggttcgac catgcgcaag cgccgccagc
     1681 gggtgcgcga ggcgttgccg gagctggtcg cgctgggctg gacggtaacc gagttcgcgg
     1741 cgggcaagta cgacatcacc cggcccaagg cggcaggctg aggccggcct acggccagcc
     1801 tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag tgaagaagga
     1861 acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc gttggataca
     1921 ccaaggaaag tctacacgaa ccctttggca aaatcctgta tatcgtgcga aaaaggatgg
     1981 atataccgaa aaaatcgcta taatgacccc gaagcagggt tatgcagcgg aaaaggacaa
     2041 cgcgcggacc gcggtccaat taattattag aaaaattcat ccagcatcag atgaaattgc
     2101 agtttgttca tatccggatt atcaatgcca tatttctgaa acagacgttt ttgcaggctc
     2161 gggctaaatt cgcccaggca gttccacaga atggccagat cctgataacg atccgcaatg
     2221 cccacacggc ccacatcaat gcagccaatc agtttgcctt catcgaaaat caggttatcc
     2281 aggctaaaat cgccgtgggt caccacgcta tccgggctaa acggcagcag tttatgcatt
     2341 tctttccaca cctgttccac cggccagccg ttacgttcat catcaaaatc gctcgcatcc
     2401 accaggccgt tgttcatacg gctctgcgcc tgggccagac gaaacacacg atcgctgtta
     2461 aacgggcagt tgcacaccgg aatgctatgc agacgacgca gaaacacggc cagcgcatcc
     2521 acaatgtttt cgccgctatc cggatattct tccagcacct gaaacgcggt tttgcccgga
     2581 atcgcggtgg tcagcagcca cgcatcatcc ggggtgcgaa taaaatgttt aatggtcggc
     2641 agcggcataa attcggtcag ccagttcaga cgcaccattt catcggtcac atcgttcgcc
     2701 acgctgcctt tgccatgttt cagaaacagt tccggcgcat ccggtttgcc atacagacga
     2761 taaatggtcg cgccgctctg acccacgtta tcacgcgccc atttatagcc atacagatcc
     2821 gcatccatgt tgctgttcag acgcggacgg ctacagctcg tttcacgctg aatatggctc
     2881 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata
     2941 tttttatctt gtgcaatgta acatcagaga ttttgagaca caaatttaaa tcgtaattat
     3001 tggggacccc tggattctca ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca
     3061 aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca agactagtcg
     3121 ccagggtttt cccagtcacg acgcggccgc aagcttgcat gcctgcaggt cgactctaga
     3181 tcagaacgga atgtcatcat caaagtccat cggcggctcg ttagacggcg ctgccggagc
     3241 ggactgctgc gggcgagact gcgcgccgcc gctgaactga ttgccaccct gcggctgctg
     3301 aggctgaccc caaccgccct gcggctgacc accaccgata ttaccacctg ccggagcgcc
     3361 accaccctga cgaccaccca gcatctgcat ggtgccgcca acgttcacca cgacttctgt
     3421 ggtgtagcga tcctgaccgg attgatcggt ccatttacgg gtacgcagct gaccttcgat
     3481 ataaacctga gaacctttac gcagatattc gctggccact tctgccagtt tgccgaacag
     3541 cacaacgcgg tgccattcag tctgctcttt catctcgccg gtcgctttat cacgccagga
     3601 ttcggaagta gccagcgtaa tgttggcaac tgcgccacca tttggcatgt agcgtacttc
     3661 cgggtcctga cccagattac caacgagaat aaccttgttt acgcctctgc tggccatgtg
     3721 tattctcctg gatccttaaa acacctcgcc ctcgtcaaca acctcatact ccgcttccac
     3781 agctggcgca gccatttctt cctccatcca agcgcgagca cggcgagctt gctgctcggt
     3841 catagagtgc atgccttcag cgcccaccgc accacaaaca gcagtcatac cctcagccgg
     3901 agtgatgccg aagtgttcgc aatatggctt gaagagttcg cggattggct ggagatccac
     3961 tggttgctcc gcctgagcgc gcacctgttc tggctgttgc gcttgatcca tttcctccgc
     4021 agcgtacatg ccctgaaaag tgtccgggaa cgcgaggcgc caagcagcag ccttcgcgca
     4081 tttttcgatc ataacacccg gcattttcgc ccaattagat ttgccggtgg aatagtcgtc
     4141 gagcgccacc tcagcatacg cagtctcgcg gccatcctta aagcgaactt ccgcccaacc
     4201 accgatgagc tcctcacccg cctttttgta gcacgcagca ccacgtttat gcacaacatc
     4261 accgtcacgc agaacaacca caccagattt gataccatca tagttagcat cacgacacgc
     4321 gcgacggtta aaaacttgat aggaagtaat catagaagct ggagcaccgc cgtatttcac
     4381 caggtaagcg tctttcacaa acggattcag acgttgagtc tgacagagtt gcagaaagag
     4441 gcccacctct ttttcagtag cgttctcaca gatataacgg cgaacatcgt ctggagtaac
     4501 ttgaaccgcc aggccagagt cgtcagtgaa cttcacgatt tggttcattc caaacctcct
     4561 taaagttaaa caaaattatt tcgatcagat tccgtatgcg cacccttacg tcagaaaaaa
     4621 cgggtttcct gaattcgggt gaagagctat tcgtaaggga caggttgaag ctacaacggc
     4681 tcacactcgc tcttcacttg aattcaggaa aacagacagt aactcagatt caatgcagga
     4741 tgccgaaaca acatccagag gttgacctta atgataaacc tctcgctaag ggtgcgcata
     4801 cggaatctta tcatagttaa atgttaggtc ttcgctttat ttaaagggtt ctttccatat
     4861 tttttttcta atttgctaat ataagttatt aatctcctat ggctttttga atccacactt
     4921 aaaataaatg acaaccatcc cctaacgtgt tctatctcag aagacttacc gcaaaatata
     4981 tgatgtatct ttgctctaat ttctttatat ttttctctac ctatcccaac tttctcttgt
     5041 gaaataacta aacctgtaac tttcctctga ctacgaggcc cactaataca agtttttttt
     5101 gagttaataa ccaatccttc acttgggatt atagaaaata aaaaatcacg tgctttaaca
     5161 acctttttca tagactgtgc agataaggtg agatcatcgg catatctcgt atatatcaag
     5221 ccccgactac ctgcataacc ctgaatacga taatcaagtt tagaacatat tagattagct
     5281 aatttaggtg atgatggagc accttgtggt agcagatttt tataacaaca tatttttgtc
     5341 aaaactgaag atattagtcg attataacca agagaatgga acactccaaa aactttgtta
     5401 gcagttaaac ttgggaaaaa atcctccaaa tcaatattca gtataaagtt tgccccaata
     5461 tgcggggtag cattattcaa aatagattgg tgcttttcaa atccaataga aaaaggagat
     5521 gacgacagtt tatctaaaat gttacgtaga acccatcctt gtaaggcttt aagttctcga
     5581 gaaggttggt aaatggttct cattctcttc tctgggcctt tcttttctac agtgtagatc
     5641 ctatagcgaa aatcagctgt atagattaac aaccgaagtg tttcaacaga tatgcgagtc
     5701 gccttagaca tgtcatgcaa attgttcatg acaggtaggc cgagatttct caatctaaaa
     5761 gtgttcaaat attcagcgga tttcatatgt atatctcctt cttaaagtta aacaaaatta
     5821 tttggctagc attgtaccaa gggctgagct agctataaag gcttgattac gaacgtttaa
     5881 ttaaaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     5941 ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgccctaga cagctgggcg
     6001 cgccctaact gtcacgaacc cctgcaataa ctgtcacgcc cccctgcaat aactgtcacg
     6061 aacccctgca ataactgtca cgcccccctg caataactgt cacgaacccc tgcaataact
     6121 gtcacgcccc caaacctgca aacccagcag gggcgggggc tggcggggtg ttggaaaaat
     6181 ccatccatga ttatctaaga ataatccact aggcgcggtt atcagcgccc ttgtggggcg
     6241 ctgctgccct tgcccaatat gcccggccag aggccggata gctggtctat tcgctgcgct
     6301 aggctacaca ccgccccacc gctgcgcggc agggggaaag gcgggcaaag cccgctaaac
     6361 cccacaccaa accccgcaga aatacgctgg agcgctttta gccgctttag cggcctttcc
     6421 ccctacccga agggtggggg cgcgtgtgca gccccgcagg gcctgtctcg gtcgatcatt
     6481 cagcccggct catccttcgg atctagctac actttatgct tccggctcgt ataatgtgtg
     6541 ggaatcgctt ggtggcccga tgaagaacga caggactttg caggccatag gccgacagct
     6601 caaggccatg ggctgtgagc gcttcgatat cggcgtcagg gacgccacca ccggccagat
     6661 gatgaaccgg gaatggtcag ccgccgaagt gctccagaac acgccatggc tcaagcggat
     6721 gaatgcccag ggcaatgacg tgtatatcag gcccgccgag caggagcggc atggtctggt
     6781 gctggtggac gacctcagcg agtttgacct ggatgacatg aaagccgagg gccgggagcc
     6841 tgccctggta gtggaaacca gcccgaagaa ctatcaggca tgggtcaagg tggccgacgc
     6901 cgcaggcggt gaacttcggg ggcagattgc ccggacgctg gccagcgagt acgacgccga
     6961 cccggccagc gccgacagcc gccactatgg ccgcttggcg ggcttcacca accgcaagga
     7021 caagcacacc acccgcgccg gttatcagcc gtgggtgctg ctgcgtgaat ccaagggcaa
     7081 gaccgccacc gctggcccgg cgctggtgca gcaggctggc cagcagatcg agcaggccca
     7141 gcggcagcag gagaaggccc gcaggctggc cagcctcgaa ctgcccgagc ggcagcttag
     7201 ccgccaccgg cgcacggcgc tggacgagta ccgcagcgag atggccgggc tggtcaagcg
     7261 cttcggtgat gacctcagca agtgcgactt tatcgccgcg cagaagctgg ccagccgggg
     7321 ccgcagtgcc gaggaaatcg gcaaggccat ggccgaggcc agcccagcgc tggcagagcg
     7381 caagcccggc cacgaagcgg attacatcga gcgcaccgtc agcaaggtca tgggtctgcc
     7441 cagcgtccag cttgcgcggg ccgagctggc acgggcaccg gcaccccgcc agcgaggcat
     7501 ggacaggggc gggccagatt tcagcatgta gtgcttgcgt tggtactcac gcctgttata
     7561 ctatgagtac tcacgcacag aagggggttt tatggaatac gaaaaaagcg cttcagggtc
     7621 ggtctacctg atcaaaagtg acaagggcta ttggttgccc ggtggctttg gttatacgtc
     7681 aaacaaggcc gaggctggcc gcttttcagt cgctgatatg gccagcctta accttgacgg
     7741 ctgcaccttg tccttgttcc gcgaagacaa gcctttcggc cccggcaagt ttctcggtga
     7801 ctgatatgaa agaccaaaag gacaagcaga ccggcgacct gctggccagc cctgacgctg
     7861 tacgccaagc gcgatatgcc gagcgcatga aggccaaagg gatgcgtcag cgcaagttct
     7921 ggctgaccga cgacgaat
//
