LOCUS       sequence-441219-        6381 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pNP1723 - IS621 WT Donor and bridgeRNA in trans (TBL-T5, DBL-WT).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6381)
  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 6381)
  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"; doi: 
            "10.1038/s41586-024-07552-4"; journalName: "Nature"; date: 
            "2024-06"; volume: "630"; issue: "8018"; pages: "984-993"
COMMENT     Sequence Label: pNP1954
FEATURES             Location/Qualifiers
     source          1..6381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(809..1891)
                     /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     1853..1872
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1892..1969)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2175..2194
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     ncRNA           complement(2325..2392)
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     promoter        complement(2570..2604)
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     terminator      complement(2712..2739)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2755..2826)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2847..3560)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     RBS             3567..3578
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     rep_origin      3691..4146
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3778..3797)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3988..4009
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4238..5053)
                     /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(4238..5053)
                     /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     4961..4980
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5045..5064
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5175..5763
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5664..5683
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5917..5934
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5949..6088
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6014..6033)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6174..6196
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6190..6381)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
       61 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
      121 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
      181 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
      241 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
      301 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
      361 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
      421 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
      481 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
      541 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
      601 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
      661 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
      721 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
      781 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
      841 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
      901 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
      961 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     1021 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     1081 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     1141 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     1201 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     1261 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     1321 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     1381 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     1441 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     1501 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     1561 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     1621 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     1681 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     1741 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     1801 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     1861 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     1921 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     1981 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     2041 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     2101 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     2161 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     2221 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga taagaaggag atataccatg
     2281 gaaaaaaaag cccgctcatt aggcgggctg acagctgtca gatcgtccca ttcgccatgc
     2341 cgaagcatgt tgcccagccg gcgccagcga ggaggctggg accatgccgg ccagtgaaac
     2401 caaccttgca aatacggatt accgggaaaa ccggtccatg atactgtccg gtttatcact
     2461 ttgggagaaa ggcagtccgc tgcataaatc taggcaacag gttagaaccc aaggggtccc
     2521 acggcatgcg gactgcaggc agagaaaact ggccgatttt ctctgcactg ctagcattat
     2581 acctaggact gagctagctg tcaacgagct cggcgcgcct gcaggtcgac aagcttgcgg
     2641 ccgcataatg cttaagtcga acagaaagta atcgtattgt acacggccgc ataatcgaaa
     2701 ttctatataa acgcagaaag gcccacccga aggtgagcca gtgtgactct agtagagagc
     2761 gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt
     2821 tatttgatgc ctggagatcc ttatcatttg tacagttcat ccataccatg cgtgatgccc
     2881 gctgcggtta cgaactccag cagaaccata tgatcgcgtt tctcgttcgg atctttagac
     2941 agaacgcttt gcgtgctcag atagtgattg tctggcagca gaacaggacc atcaccgatt
     3001 ggagtgtttt gctggtagtg atcagccagc tgcacgctgc catcctccac gttgtggcga
     3061 attttaaaat tcgctttaat gccatttttt tgtttatcgg cggtgatgta aacattgtgg
     3121 ctgttaaaat tgtattccag cttatggccc aggatattgc cgtcttcttt aaagtcaatg
     3181 cctttcagct caatgcggtt taccagggta tcgccttcaa atttcacttc cgcacgcgtt
     3241 ttgtacgtgc cgtcatcctt aaaggaaatc gtgcgttcct gcacatagcc ttccggcatg
     3301 gcggacttga agaagtcatg ctgcttcata tggtccggat aacgagcaaa gcactgaaca
     3361 ccataagtca gcgtcgttac cagagtcggc caaggaaccg gcagtttacc agtagtacag
     3421 atgaacttca gcgtcagttt accattagtt gcgtcacctt caccctcgcc acgcacggaa
     3481 aacttatgac cgttgacatc accatccagt tccaccagaa tagggacgac accagtgaac
     3541 agctcttcgc ctttacgcat agatcatttc tcctctttca gatccggacc tcctaaatag
     3601 ttagtttgac gcaccggata acagtatctt gtattatccg gatctcgagc accaccaaaa
     3661 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     3721 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     3781 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     3841 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     3901 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     3961 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4021 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     4081 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     4141 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4201 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     4261 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     4321 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     4381 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     4441 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     4501 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     4561 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     4621 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     4681 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     4741 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     4801 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     4861 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     4921 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     4981 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     5041 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     5101 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5161 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5221 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5281 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     5341 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5401 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5461 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5521 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5581 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5641 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5701 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5761 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5821 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5881 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5941 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     6001 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     6061 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     6121 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     6181 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     6241 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     6301 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     6361 ggttttttcc tgtttggtca c
//
