LOCUS       sequence-495450-        4850 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  Rep101 Ts - IS621 WT Donor (22bp) with Bba_R0040-KanR Cargo and 
            Rep101 Temp Sensitive.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4850)
  AUTHORS   Durrant MG, Perry NT, Pai JJ, Jangid AR, Athukoralage JS, Hiraizumi 
            M, McSpedon JP, Pawluk A, Nishimasu H, Konermann S, Hsu PD
  TITLE     Bridge RNAs direct programmable recombination of target and donor 
            DNA.
  JOURNAL   Nature. 2024 Jun;630(8018):984-993. doi: 10.1038/s41586-024-07552-4.
            Epub 2024 Jun 26.
  PUBMED    38926615
REFERENCE   2  (bases 1 to 4850)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-024-07552-4"; journalName: "Nature"; date: 
            "2024-06"; volume: "630"; issue: "8018"; pages: "984-993"
COMMENT     Sequence Label: pNP2085
FEATURES             Location/Qualifiers
     source          1..4850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..223
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     primer_bind     806..825
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(970..997)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(1013..1084)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1102..1917)
                     /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"
     CDS             complement(1102..1917)
                     /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
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1825..1844
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     RBS             1924..1935
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    1953..1971
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1978..1996
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(2146..2165)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2371..2448
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2449..3531
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2468..2487)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             complement(3853..4803)
                     /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"
ORIGIN
        1 gggctagtca atgataatta ctagtccttt tcctttgagt tgtgggtatc tgtaaattct
       61 gctagacctt tgctggaaaa cttgtaaatt ctgctagacc ctctgtaaat tccgctagac
      121 ctttgtgtgt tttttttgtt tatattcaag tggttataat ttatagaata aagaaagaat
      181 aaaaaaagat aaaaagaata gatcccagcc ctgtgtataa ctcactactt tagtcagttc
      241 cgcagtatta caaaaggatg tcgcaaacgc tgtttgctcc tctacaaaac agaccttaaa
      301 accctaaagg cttaagtagc accctcgcaa gctcgggcaa atcgctgaat attccttttg
      361 tctccgacca tcaggcacct gagtcgctgt ctttttcgtg acattcagtt cgctgcgctc
      421 acggctctgg cagtgaatgg gggtaaatgg cactacaggc gccttttatg gattcatgca
      481 aggaaactac ccataataca agaaaagccc gtcacgggct tctcagggcg ttttatggcg
      541 ggtctgctat gtggtgctat ctgacttttt gctgttcagc agttcctgcc ctctgatttt
      601 ccagtctgac cacttcggat tatcccgtga caggtcattc agactggcta atgcacccag
      661 taaggcagcg gtatcatcaa ccaggtggca cttttcgggg aaatgtgttg gggtcgaggt
      721 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
      781 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
      841 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
      901 tacagggcgc gtcccattcg ccaatccgga tatagttcct ccttttggtg gtgctcgagt
      961 ctatataaac gcagaaaggc ccacccgaag gtgagccagt gtgactctag tagagagcgt
     1021 tcaccgacaa acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta
     1081 tttgatgcct ggagatcctt attagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     1141 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     1201 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     1261 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     1321 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     1381 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     1441 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     1501 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     1561 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     1621 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     1681 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     1741 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     1801 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     1861 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataga
     1921 tcatttctcc tctttcagat ccgtgctcag tatctctatc actgataggg atgtcaatct
     1981 ctatcactga tagggaggac ctcctaaata gttagtttga cgcacataat aacagtatct
     2041 tgtattatcc ggatgtgccc atggtatatc tccttcttat ctcgatcctc tacgccggac
     2101 gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca
     2161 tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg
     2221 gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat
     2281 tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg
     2341 agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc
     2401 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta
     2461 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     2521 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     2581 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     2641 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     2701 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     2761 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     2821 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     2881 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa
     2941 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     3001 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat
     3061 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc
     3121 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
     3181 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt
     3241 ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg
     3301 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
     3361 ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg
     3421 aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     3481 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     3541 aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc
     3601 cttcaaccca gtcagctcct tggataagag acggctcaac gcccgtctca cagcgcaata
     3661 tcaatttatg ggtgtgataa ttatcaattt atgggtgtaa ttatcatttt atggttgtat
     3721 caacaaggag gactttggca ggtgcctgca ggtatggaca gttttccctt tgatatgtaa
     3781 cggtgaacag ttgttctact tttgtttgtt agtcttgatg cttcactgat agatacaaga
     3841 gccataagaa cctcagatcc ttccgtattt agccagtatg ttctctagtg tggttcgttg
     3901 tttttgcgtg agccatgaga acgaaccatt gagatcatac ttactttgca tgtcactcaa
     3961 aaattttgcc tcaaaactgg tgagctgaat ttttgcagtt aaagcatcgt gtagtgtttt
     4021 tcttagtccg ttatgtaggt aggaatctga tgtaatggtt gttggtattt tgtcaccatt
     4081 catttttatc tggttgttct caagttcggt tacgagatcc atttgtctat ctagttcaac
     4141 ttggaaaatc aacgtatcag tcgggcggcc tcgcttatca accaccaatt tcatattgct
     4201 gtaagtgttt aaatctttac ttattggttt caaaacccat tggttaagcc ttttaaactc
     4261 atggtagtta ttttcaagca ttaacatgaa cttaaattca tcaaggctaa tctctatatt
     4321 tgccttgtga gttttctttt gtgttagttc ttttaataac cactcataaa tcctcataga
     4381 gtatttgttt tcaaaagact taacatgttc cagattatat tttatgaatt tttttaactg
     4441 gaaaagataa ggcaatatct cttcactaaa aactaattct aatttttcgc ttgagaactt
     4501 ggcatagttt gtccactgga aaatctcaaa gcctttaacc aaaggattcc tgatttccac
     4561 agttctcgtc atcagctctc tggttgcttt agctaataca ccataagcat tttccctact
     4621 gatgttcatc atctgaacgt attggttata agtgaacgat accgtccgtt ctttccttgt
     4681 agggttttca atcgtggggt tgagtagtgc cacacagcat aaaattagct tggtttcatg
     4741 ctccgttaag tcatagcgac taatcgctag ttcatttgct ttgaaaacaa ctaattcaga
     4801 catacatctc aattggtcta ggtgatttta atcactatac caattgagat
//
