LOCUS       sequence-482476-        9106 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pLAM12 vector backbone containing Ebu1 recombitron with MspRecT and 
            a donor in the ncRNA to target MSMEG_5894 gene of Mycobacterium 
            smegmatis mc2 155.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9106)
  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 9106)
  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: pRECMYC-Ebu1
FEATURES             Location/Qualifiers
     source          1..9106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(3927..4515)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4007..4026)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4846..5661)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5569..5588
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5653..5672
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      complement(5822..5868)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gcggtgaatg tcgtcagccc taccgaaccg aagacccggc gcgccggcgt ccggcactcc
       61 ggacaggtcg ccgccgcatc gcgctcggag atcgcgcagg tgagatcgaa gggaccgcag
      121 tgggaacaac ggaatgtgta ggtcggcatg gtcacccctt tccattcacc cgaaatatat
      181 ccagatgaaa ataaaagtca agggtgaacg cccgcgccgg gcgcgggtgg ttacgaagtc
      241 aaattcgagg tggtgtcgac gagttcgcgc aactggtcga cgtccagccc gcacgattca
      301 aaagccgcac actcggcctc ggctaccgcg tccaccaggc ggttgcgcag cgtccgcccc
      361 cgcggggtca acgccacgag cacccgccgg cggtcatgca gatcggcagt ccggtagatg
      421 atcccttgcg acacaaggcg atcgacggtt ctggtgagag tcggacccgt gatcagcgtg
      481 gcctgcgcga gatcgctcat gggcacggtg cgccgtccgg ccaactcgtc gagcacgcgc
      541 cagtcctcga tcttgagcga cgagtccgcg agcacgcgtt cgatcgccga ctccgcgagg
      601 tgggtcagac gcaccaacgg cagcaggagg ttggggcggc gctgctgtcc ggcggccatg
      661 gtgggtgtct cctcgtgtcg acggcggggt ctgttcagaa tgaattattt ctgctagaaa
      721 gctttgaagc gcagactaca cgggatcgac ggtgatctgg agtgcaggac ggcgaggttg
      781 agttccgcgt cgggctggtg attccgcttc aagggccagc gggaatcttc gcgccttcgt
      841 gcgaggccgt ggccgagctc gcggccaagg aggtcaacga ccgaggcggc ctgcagggcc
      901 gcaaggtcac catcgaggtg ctcgacggcg gagcgcccgg cgacgacgtc gcccggaccg
      961 tcgccgaccg gttgcgaggt cacggtctcg acgccgtgac cggctggcac atctcggccg
     1021 tgcgcaaccg catctccccg gtggtccgcg accgcatccc gtacgtctac acctcgttgt
     1081 acgagggcgg tgaacgcaca ccgggcgtgt tctgcacagg cgagacaccg cagatccaga
     1141 tcgcgcccgc gctcgcctgg ctgcgcgacc acttcggcat ccggtcctgg tgcctggtcg
     1201 gcgatgacta catctggccg cgccgttccg ccgcggccgc ccgcgcgtac tgccgagatc
     1261 tggacctcga gctcagacgg gagatctacg tcccgtacgg caccgacgat ttccgcgcac
     1321 ctgtccgcaa ggccatcgcg tcgggggcgc aggccgtgtt gatgctgctc gtcggccagg
     1381 acgccgtgtt gttcaaccgc gagttcgcgc gcgccggcgg gcacgaccgc atggcgcggt
     1441 tcagcccgct gatggaggag aacatgctgc tggccagcgg cgccggctcc accgaaaacc
     1501 tctatgtcgc cgcggcgtac ttcagttcac tggccaccgc gggtgccatg gacctgatgg
     1561 gcagctacgt cgcccgctac ggcgccgacg cgccaccgct caacgcgatg gccgaatcct
     1621 gttacgaggg tctgcttgcg ctcgaggcca tcttccagcg ggcccactcc cccgagatac
     1681 cggacctgat ggcatccgcg cacgatgtcg ggttcgacgg gccccggggc ccgatgtgta
     1741 tgcgcgacag tcaattcgac cagcaggtct acatcgccag cgccgacggc tacgacttcg
     1801 acatcctcga cacgctggcg acgctcgacg cttgagaccg cgtcacttct ttatctagat
     1861 ttaaagatct ggtaccgcgg ccgcgccacg gatgccacca caggcactac aacggagttc
     1921 gccacgtaca tcaccacaac caccgattct ggcggtgagc tccccgatat tcagcggaaa
     1981 tggcttggta tcgaccaaga ttcgtagaac cccgtctcgt ctggctggta ttcaaaacgg
     2041 gcgcaacgaa acacgcaacg agacaggcat ggcccaaacc agaaaactag cgtctaccag
     2101 gacttttacc tgtccgaccc gttgcaacgg aaccccccac ggaacccccg cgacacccgc
     2161 tccccaattg cgttagaaca gcggtggatt gtcggcttcg ttgtgggcct tttgagccgc
     2221 ttcctgttct gccgcacgct ctttcctcgc ccgatagccg agtcgcttaa cggtgtccag
     2281 atgcagcccg aaatgtttgg ccgtttgcgg ccaagagtgg ccctcgtcgt cgtgataggc
     2341 gcggatgcgt tcgcggcgtg cagcctgctc ggcgagccac tcgctgcgtt cctgcgccac
     2401 gagccggacg acgtggcgtt cggatagtcc ggtgattcga gcgccttcgg cggcggtcac
     2461 gcgccgcttt ttgcggacag tcggctgccg gttgtagccg tcgctgtagc cgtcgctcat
     2521 agcaatgcct ccatggctga cgcggacttt gcgcgccgcg caactgtgct cgccgccgtg
     2581 cgcgctgctg cgcccttccg cgagatggcc gactggcgcg cactgagtgt ggcctcgtag
     2641 accacgatcc cgtccgccca aatgcgcgac ttggttgtga tccaacgcca aatgctgttg
     2701 gcgatggcgc ggacctcgct gtccggtagc ggtccgggac acacgtcgtt gcacggaaat
     2761 tcggcgtttc gcgcgtggca ctcggcatag atcgcgcggc cgagtccgtc cacgttccgg
     2821 gtcggcaggt agatccgcat gagggcggga cgataggccc acaacctgac ggaatcgaac
     2881 agtgcgcaat tccgccctag cggcgtcgga gccgctttgt acgtggtctg ctgacgccag
     2941 cgcggcggtg gcatgttcgc gccgagctcg gcctcgatgt ggctgagtgt gtagagatct
     3001 gagtggagcc attccgtttc ccaggcgatg tggccggggt ttttggtcat gaggcctgag
     3061 taactgcggt cgccatcgac ggcgcgccga aggccttcgg cgcacgccgc catgtatgcg
     3121 agcggcttac gccgcgcgta ttcggtgcgt ggaacagggg cgttgagtgc ccacactgcg
     3181 tgtgcgtggc cgttggcgcg attgcccacg atcgcgttgg gcagcggatg ggacccccgg
     3241 gcgctgagcg ctcggagcgc tgcgtctgga tggtctacgt ccacgaccag caggtttgcc
     3301 agcgctgttg ggttcgcctc gatgtaccgg cggcctaggg ccgacgcgcg gctttggcgg
     3361 tagatcccct cgagcagatc gtcgcttgcc agcggccagt acggcagcca gagctgctca
     3421 aattcgtcgg cgacgtggct cacgcttggt agtagaccac gattaatcac cggtgtatgg
     3481 tccgacacga gctccaagtc agatatttcg ctgaggggcc accccacaac tgcacactcc
     3541 cccgctctcc cgtcgagccc tggtggtgga acaccagcga cagccgagca cccccaacca
     3601 cctgtaccaa ccaggaggaa cacatgcgtc gtttcgagga cgtttccggg ccgctgagag
     3661 ccgctgtggc ggccgtacac gccgccttag acccgttaga ccccctgccg cctgaatgcg
     3721 cgggtacgag ccacacagcg cccgaactta cggagctggt gggctcacgc gttgcgctcg
     3781 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3841 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3901 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3961 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4021 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4081 ctgtccgcct ttctcccctc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4141 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4201 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4261 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4321 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     4381 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4441 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4501 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcatggaact
     4561 agtgaggtct gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga atcgccccat
     4621 catccagcca gaaagtgagg gagccacggt tggtgagagc tttgttgtag gtggaccagt
     4681 tggtgatttt gaacttttgc tttgccacgg aacggtctgc gttgtcggga agatgcgtga
     4741 tctgatcctt caactcagca aaagttcgat ttattcaaca aagccgccgt cccgtcaagt
     4801 cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgattaga aaaactcatc
     4861 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     4921 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     4981 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     5041 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     5101 tggcaaaaga ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     5161 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     5221 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     5281 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     5341 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     5401 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     5461 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatg
     5521 gggcttccca tacaagcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     5581 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg agcaagacgt
     5641 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     5701 tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac
     5761 aacgtggctt tccccccccc ccgacgtcag gtggctagct gatcaccgcg gccatgatgg
     5821 cataaaacga aaggcccagt ctttcgactg agcctttcgt tttatttgat gcctggcagt
     5881 cgatcgtacg ctagttgcag atcaaccacc gcgtcggaat tcgccggggc gctcatgcgt
     5941 tgggcccgtc gaacatgtcg atgacagcct tcgcctcgtc gaacgtgagg tcggcggcgc
     6001 gcgtggcttt gacgccggcc gaatcggcga ggaacttgaa ccaatcggcg tcgttgtact
     6061 tttcggcctt ctggatctcg ccgaggcgct tcagctgatc gggattggcc atctgcacgt
     6121 cgggcgccgg cgcggtggcg cgctgcgcgc gttgtgcgcg cgacgacttc ggtggcgtat
     6181 cgacctcgtg cggctcggcg atctcgccgt cgatgtagtc ggccgtcggc ttgacggtcg
     6241 acggcagcgc gagcgcctgc gactgtgaca ggtcggtgcc cgggcgatcg tcggcgcgga
     6301 tcgccgcgtc gagccgcgtc gtcttcggtg ccagcttcag cacctgtttg agcgcggtct
     6361 tccgctccat ccagcgctgc gggtccttga tgtcgcccga ggatccgacc ttgccgcgac
     6421 gcaattccct gatctcgtcg gcggtgaaca cgtcccacag cggctcagcg ccggtcacct
     6481 cgacgatcgc gtagaagtag accggattac cccgatcacc cttggccttc acgtgtttca
     6541 gcgtcggatt gaggcccatg gtgtaatgga attcgtcgtt cgcgccgacc cactgcgcgt
     6601 cgatccggga ggcgcgcggg tgctgccaga acagtttgac gatgccctgg tagccgatga
     6661 tcagctggca ctcgaccacg ccgcgccggg tgtcgcggta gggcacgagg tagcactcgc
     6721 cgttgacacc gggctcgaga ccgagcgcgg ccgaggtcag cagcgcaccg gcaaatgact
     6781 cttgcgtgca gtcgtcgagg gactgcttag cgatgccagc agctttcgcg ttgcgcgact
     6841 tgcggatctc ggtttgcacg atccgcgcga tgcggtcggg gtccataccc ttgggcactg
     6901 cacgcgccag ctgcggcacg agcacctgca gcacttgcga gatcgtctcg ggtgccttgg
     6961 gcgaatcctg cttggtgaca gcattttcag ccatgattgg ctttctcctt tctcccgccg
     7021 ggatcatgca ggtggcgact tgtagaaact actttctata cctgacacct aagttctctc
     7081 ctctggcgcc ggggtgatgg tgtccagcgg ctcacacggg ccccgccggg cgtgacgcac
     7141 gacggcgcaa aagaaccgcg agaatagcaa cgctattcga agcgactata cggagttatc
     7201 cgtatagtcg tggttaaagt gacccttaac caaggagcga ctacactcca acgtgctagt
     7261 ttctacaagt catcacctgc tctagcctca tatgaacggt attatttcat ttggttgatg
     7321 tgctcaatat aggagcgcgc ttcctgattg tccggctcca cctgcaacca gaaacggatc
     7381 ttgccttcca gggatttgat gtaattcggg tctttaattt tttcgttgtc aatgtgggaa
     7441 aagatcccat atttcttaat ataatgcatc gtcttccgga tctcacgccg ggttttacgc
     7501 ggcagttgga gcttgtcctt cgacacatcg atgcccgtca cgatcttctg cttggtgtta
     7561 ttcaacaatt tggttttctc cttattgagg ataaagccat acgattcgat aatatcggtg
     7621 acgtacgaga tgaatttgga ggggatcgct tcaccggaaa agaccatgtc gtccgcatac
     7681 cgggagtact tcagattgaa cttcagcgcg agtttgctca ggcgctcatc caagggatac
     7741 aacaagatgt tggacaggct cggggaggta atgccaccct ggggcaaaca gccatccaga
     7801 cagcacaggc tcgccaacgc gtaggacaca tttttggcgt agcccaggct atcgaacaat
     7861 ttaatcaccc agttaatcgg gatgcttgga aaaaagtctt tgatatcaat tttgagcaga
     7921 aagtgattgt tcaaatgttg ttccgcgttg tcgataatgg atttcccttt tttgtacgca
     7981 aacgcgtttt tatgggtccg caaggaatac aagagcttct cgtgaatcca ggtttgacac
     8041 ttacggagcg agggatatgg ggacgaaatt ttacgatagc cgcccttttt ctttgggatt
     8101 ttaaactccc ggtaaaagga tttggtgccg aagacaatgt cattgatttc cggatatttg
     8161 atgccgatca gcttgctgag gtgttcgact tcaaagatga ctgggatgtt ccgcgactcc
     8221 aaggttttaa tataatccaa gtaggtggaa atgaagttcg tattgatccc cttttccaag
     8281 tagaagtcct cccactcccg ccgcatatgt ggactccctt tctcttatcg ggtggtggcc
     8341 gcgagcgcgg ccggtggagg gttggtgact gacggcgctg cgagacggcg cccgatgggg
     8401 tatgcgagaa caactgcggc aaaaccgatc acggcgatga ccgcggcggg gagcgggccg
     8461 gtttcccagt tgcccgacac gatcaggaag gccggcacgg cggcggtgat cacgccttcg
     8521 gccaccgcga cgaacccgac ggcgggcccc agtgcgtcgt gccccaggcc gagcagcagg
     8581 aacagcatga accacagcac tgcccacagc agccagatca ccccgaacgc cgggtcggcc
     8641 tcggcggtga acgcgtgcca cgagtagccg agtgcggcga tcgcgacgaa cagcgagaac
     8701 catccgagac cttctccgtc ccagtcggtc acgttgttga tggccaccca caggtaggtg
     8761 aagccgaaca ggtagagccc ggaggccgcg aagatcacgg ccgcgtctcc gccggactgc
     8821 aggatcagca ccgtaggcgt caccacctgc agtccgccga cgaagaagtt cagcggtgtt
     8881 gcgcctcgtg ggctgatgcg gcccagcagc atcagcccgt cgatgatgag caccgcaccc
     8941 acgtagaaga gcccgacgcc acccatgtcg ctagtacctc ggcagactcg gtagaccggg
     9001 attgcggcgc ggggcccgcg ggcggtccgc gcctgcggga atcgacttca ccaccgtggg
     9061 actttcggcg ctcgcggacg ccgcgtcgaa tgcgcggtga tgtccc
//
