LOCUS       sequence-440101-        5938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sfGFP-ON BsaI Golden Gate Landing Pad Backbone for RE-LE (bRNA in 
            cis expression).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5938)
  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 5938)
  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: pNP555
FEATURES             Location/Qualifiers
     source          1..5938
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..192
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(186..208)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    294..433
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     349..368
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(448..465)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(619..1207)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(699..718)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1318..1337)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1329..2144
                     /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             1329..2144
                     /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     complement(1402..1421)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2236..2691)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2373..2394)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2585..2604
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2749..2776)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2792..2863)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2884..3597)
                     /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             3604..3615
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(3745..3764)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3970..4047
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4048..5130
                     /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(4067..4086)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta
       61 acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg
      121 cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt
      181 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
      241 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
      301 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
      361 agattgtact gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa
      421 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
      481 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
      541 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
      601 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
      661 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
      721 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
      781 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
      841 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
      901 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
      961 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     1021 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     1081 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     1141 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     1201 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     1261 acgttaaggg attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     1321 ggggtgttat gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca
     1381 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     1441 cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     1501 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     1561 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     1621 ccactgcgat ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     1681 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     1741 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     1801 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     1861 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     1921 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     1981 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     2041 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     2101 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc
     2161 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     2221 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     2281 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     2341 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     2401 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     2461 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     2521 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     2581 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     2641 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     2701 caatccggat atagttcctc cttttggtgg tgctcgagtc tatataaacg cagaaaggcc
     2761 cacccgaagg tgagccagtg tgactctagt agagagcgtt caccgacaaa caacagataa
     2821 aacgaaaggc ccagtctttc gactgagcct ttcgttttat ttgatgcctg gagatcctta
     2881 tcatttgtac agttcatcca taccatgcgt gatgcccgct gcggttacga actccagcag
     2941 aaccatatga tcgcgtttct cgttcggatc tttagacaga acgctttgcg tgctcagata
     3001 gtgattgtct ggcagcagaa caggaccatc accgattgga gtgttttgct ggtagtgatc
     3061 agccagctgc acgctgccat cctccacgtt gtggcgaatt ttaaaattcg ctttaatgcc
     3121 atttttttgt ttatcggcgg tgatgtaaac attgtggctg ttaaaattgt attccagctt
     3181 atggcccagg atattgccgt cttctttaaa gtcaatgcct ttcagctcaa tgcggtttac
     3241 cagggtatcg ccttcaaatt tcacttccgc acgcgttttg tacgtgccgt catccttaaa
     3301 ggaaatcgtg cgttcctgca catagccttc cggcatggcg gacttgaaga agtcatgctg
     3361 cttcatatgg tccggataac gagcaaagca ctgaacacca taagtcagcg tcgttaccag
     3421 agtcggccaa ggaaccggca gtttaccagt agtacagatg aacttcagcg tcagtttacc
     3481 attagttgcg tcaccttcac cctcgccacg cacggaaaac ttatgaccgt tgacatcacc
     3541 atccagttcc accagaatag ggacgacacc agtgaacagc tcttcgcctt tacgcataga
     3601 tcatttctcc tctttcagat ccgcaccgag accatggcca ccggtctcag gatgtgccca
     3661 tggtatatct ccttcttatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg
     3721 gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg
     3781 ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg
     3841 ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca
     3901 acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc
     3961 gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
     4021 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
     4081 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     4141 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     4201 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     4261 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     4321 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     4381 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     4441 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     4501 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     4561 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     4621 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     4681 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     4741 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     4801 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     4861 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     4921 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     4981 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     5041 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     5101 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     5161 tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
     5221 ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
     5281 actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg
     5341 gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca
     5401 agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg
     5461 catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg
     5521 gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga
     5581 ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt
     5641 ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat
     5701 cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca
     5761 ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc
     5821 acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc
     5881 tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatca
//
