LOCUS       sequence-440095-        6225 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sfGFP-ON BsaI Golden Gate (GG) Landing Pad for LD-RD and J23119-BsaI
            GG Landing Pad for bRNA (bRNA in trans expression).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6225)
  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 6225)
  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: pNP1723
FEATURES             Location/Qualifiers
     source          1..6225
                     /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(2416..2450)
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     terminator      complement(2558..2585)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2601..2672)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2693..3406)
                     /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             3413..3424
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     rep_origin      3535..3990
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3622..3641)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3832..3853
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4082..4897)
                     /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(4082..4897)
                     /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     4805..4824
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4889..4908
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5019..5607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5508..5527
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5761..5778
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5793..5932
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5858..5877)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6018..6040
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6034..6225)
                     /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 cctgagaccc
     2401 gagccgaagg tctcagctag cattatacct aggactgagc tagctgtcaa cgagctcggc
     2461 gcgcctgcag gtcgacaagc ttgcggccgc ataatgctta agtcgaacag aaagtaatcg
     2521 tattgtacac ggccgcataa tcgaaattct atataaacgc agaaaggccc acccgaaggt
     2581 gagccagtgt gactctagta gagagcgttc accgacaaac aacagataaa acgaaaggcc
     2641 cagtctttcg actgagcctt tcgttttatt tgatgcctgg agatccttat catttgtaca
     2701 gttcatccat accatgcgtg atgcccgctg cggttacgaa ctccagcaga accatatgat
     2761 cgcgtttctc gttcggatct ttagacagaa cgctttgcgt gctcagatag tgattgtctg
     2821 gcagcagaac aggaccatca ccgattggag tgttttgctg gtagtgatca gccagctgca
     2881 cgctgccatc ctccacgttg tggcgaattt taaaattcgc tttaatgcca tttttttgtt
     2941 tatcggcggt gatgtaaaca ttgtggctgt taaaattgta ttccagctta tggcccagga
     3001 tattgccgtc ttctttaaag tcaatgcctt tcagctcaat gcggtttacc agggtatcgc
     3061 cttcaaattt cacttccgca cgcgttttgt acgtgccgtc atccttaaag gaaatcgtgc
     3121 gttcctgcac atagccttcc ggcatggcgg acttgaagaa gtcatgctgc ttcatatggt
     3181 ccggataacg agcaaagcac tgaacaccat aagtcagcgt cgttaccaga gtcggccaag
     3241 gaaccggcag tttaccagta gtacagatga acttcagcgt cagtttacca ttagttgcgt
     3301 caccttcacc ctcgccacgc acggaaaact tatgaccgtt gacatcacca tccagttcca
     3361 ccagaatagg gacgacacca gtgaacagct cttcgccttt acgcatagat catttctcct
     3421 ctttcagatc cggacctcct aaatagttag tttgacgcac cgagaccatg gccaccggtc
     3481 tcaggatctc gagcaccacc aaaaggagga actatatccg gattggcgaa tgggacgcgc
     3541 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3601 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3661 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3721 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3781 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3841 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3901 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3961 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     4021 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     4081 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     4141 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     4201 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     4261 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     4321 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     4381 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     4441 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     4501 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     4561 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     4621 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     4681 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     4741 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     4801 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     4861 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     4921 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4981 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5041 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5101 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5161 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5221 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5281 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5341 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5401 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5461 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5521 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5581 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5641 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5701 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5761 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     5821 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     5881 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     5941 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     6001 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     6061 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     6121 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     6181 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcac
//
