LOCUS       sequence-435233-        5648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCDF-Duet deriviative with sfGFP flanked by 2 BsaI sites downstream 
            of the first T7 promoter and independent msr and RT downstream of 
            the second T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5648)
  AUTHORS   Gonzalez-Delgado A, Lopez SC, Rojas-Montero M, Fishman CB, Shipman 
            SL
  TITLE     Simultaneous multi-site editing of individual genomes using retron 
            arrays.
  JOURNAL   Nat Chem Biol. 2024 Nov;20(11):1482-1492. doi: 
            10.1038/s41589-024-01665-7. Epub 2024 Jul 9.
  PUBMED    38982310
REFERENCE   2  (bases 1 to 5648)
  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/s41589-024-01665-7"; journalName: "Nat Chem Biol"; date: 
            "2024-11"; volume: "20"; issue: "11"; pages: "1482-1492"
COMMENT     Sequence Label: pAGD236
FEATURES             Location/Qualifiers
     source          1..5648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        51..69
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    70..94
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     primer_bind     complement(1223..1241)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1237..1284
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1455..2246)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2247..2338)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2386..3124)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(3334..4416)
                     /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     4378..4397
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4417..4494)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        4540..4558
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4559..4583
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     RBS             4760..4771
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4778..5491
                     /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"
     terminator      5503..5574
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5590..5617
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
ORIGIN
        1 tttaatcgaa cagaaagtaa tcgtattgta cacggccgca taatcgaaat taatacgact
       61 cactataggg gaattgtgag cggataacaa ttccccatct tagtatatta gttaagtata
      121 agatgcgcac ccttagcgag aggtttatca ttaaggtcaa cctctggatg ttgtttcggc
      181 atcctgcatt gaatctgagt tactgtctgt tttccttgtt ggtagaaatg ttattgaagg
      241 agatatacat atgaaatccg ctgaatattt gaacactttt agattgagaa atctcggcct
      301 acctgtcatg aacaatttgc atgacatgtc taaggcgact cgcatatctg ttgaaacact
      361 tcggttgtta atctatacag ctgattttcg ctataggatc tacactgtag aaaagaaagg
      421 cccagagaag agaatgagaa ccatttacca accttctcga gaacttaaag ccttacaagg
      481 atgggttcta cgtaacattt tagataaact gtcgtcatct cctttttcta ttggatttga
      541 aaagcaccaa tctattttga ataatgctac cccgcatatt ggggcaaact ttatactgaa
      601 tattgatttg gaggattttt tcccaagttt aactgctaac aaagtttttg gagtgttcca
      661 ttctcttggt tataatcgac taatatcttc agttttgaca aaaatatgtt gttataaaaa
      721 tctgctacca caaggtgctc catcatcacc taaattagct aatctaatat gttctaaact
      781 tgattatcgt attcagggtt atgcaggtag tcggggcttg atatatacga gatatgccga
      841 tgatctcacc ttatctgcac agtctatgaa aaaggttgtt aaagcacgtg attttttatt
      901 ttctataatc ccaagtgaag gattggttat taactcaaaa aaaacttgta ttagtgggcc
      961 tcgtagtcag aggaaagtta caggtttagt tatttcacaa gagaaagttg ggataggtag
     1021 agaaaaatat aaagaaatta gagcaaagat acatcatata ttttgcggta agtcttctga
     1081 gatagaacac gttaggggat ggttgtcatt tattttaagt gtggattcaa aaagccatag
     1141 gagattaata acttatatta gcaaattaga aaaaaaatat ggaaagaacc ctttaaataa
     1201 agcgaagacc taactgctgc caccgctgag caataactag cataacccct tggggcctct
     1261 aaacgggtct tgaggggttt tttgctgaaa cctcaggcat ttgagaagca cacggtcaca
     1321 ctgcttccgg tagtcaataa accggtaaac cagcaataga cataagcggc tatttaacga
     1381 ccctgccctg aaccgacgac cgggtcatcg tggccggatc ttgcggcccc tcggcttgaa
     1441 cgaattgtta gacattattt gccgactacc ttggtgatct cgcctttcac gtagtggaca
     1501 aattcttcca actgatctgc gcgcgaggcc aagcgatctt cttcttgtcc aagataagcc
     1561 tgtctagctt caagtatgac gggctgatac tgggccggca ggcgctccat tgcccagtcg
     1621 gcagcgacat ccttcggcgc gattttgccg gttactgcgc tgtaccaaat gcgggacaac
     1681 gtaagcacta catttcgctc atcgccagcc cagtcgggcg gcgagttcca tagcgttaag
     1741 gtttcattta gcgcctcaaa tagatcctgt tcaggaaccg gatcaaagag ttcctccgcc
     1801 gctggaccta ccaaggcaac gctatgttct cttgcttttg tcagcaagat agccagatca
     1861 atgtcgatcg tggctggctc gaagatacct gcaagaatgt cattgcgctg ccattctcca
     1921 aattgcagtt cgcgcttagc tggataacgc cacggaatga tgtcgtcgtg cacaacaatg
     1981 gtgacttcta cagcgcggag aatctcgctc tctccagggg aagccgaagt ttccaaaagg
     2041 tcgttgatca aagctcgccg cgttgtttca tcaagcctta cggtcaccgt aaccagcaaa
     2101 tcaatatcac tgtgtggctt caggccgcca tccactgcgg agccgtacaa atgtacggcc
     2161 agcaacgtcg gttcgagatg gcgctcgatg acgccaacta cctctgatag ttgagtcgat
     2221 acttcggcga tcaccgcttc cctcatactc ttcctttttc aatattattg aagcatttat
     2281 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     2341 gctagctcac tcggtcgcta cgctccgggc gtgagactgc ggcgggcgct gcggacacat
     2401 acaaagttac ccacagattc cgtggataag caggggacta acatgtgagg caaaacagca
     2461 gggccgcgcc ggtggcgttt ttccataggc tccgccctcc tgccagagtt cacataaaca
     2521 gacgcttttc cggtgcatct gtgggagccg tgaggctcaa ccatgaatct gacagtacgg
     2581 gcgaaacccg acaggactta aagatcccca ccgtttccgg cgggtcgctc cctcttgcgc
     2641 tctcctgttc cgaccctgcc gtttaccgga tacctgttcc gcctttctcc cttacgggaa
     2701 gtgtggcgct ttctcatagc tcacacactg gtatctcggc tcggtgtagg tcgttcgctc
     2761 caagctgggc tgtaagcaag aactccccgt tcagcccgac tgctgcgcct tatccggtaa
     2821 ctgttcactt gagtccaacc cggaaaagca cggtaaaacg ccactggcag cagccattgg
     2881 taactgggag ttcgcagagg atttgtttag ctaaacacgc ggttgctctt gaagtgtgcg
     2941 ccaaagtccg gctacactgg aaggacagat ttggttgctg tgctctgcga aagccagtta
     3001 ccacggttaa gcagttcccc aactgactta accttcgatc aaaccacctc cccaggtggt
     3061 tttttcgttt acagggcaaa agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3121 gatcttttct actgaaccgc tctagatttc agtgcaattt atctcttcaa atgtagcacc
     3181 tgaagtcagc cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgcccac
     3241 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     3301 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3361 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3421 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     3481 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     3541 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     3601 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     3661 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     3721 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     3781 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     3841 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     3901 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     3961 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     4021 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     4081 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     4141 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     4201 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     4261 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     4321 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     4381 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     4441 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     4501 ctcgacgctc tcccttatgc gactcctgca ttaggaaatt aatacgactc actatagggg
     4561 aattgtgagc ggataacaat tcccctgaat tcttctagag gtagagagac cgaaagtgaa
     4621 acgtgatttc atgcgtcatt ttgaacattt tgtaaatctt atttaataat gtgtgcggca
     4681 attcacattt aatttatgaa tgttttctta acatcgcggc aactcaagaa acggcaggtt
     4741 cggatcttag ctactagaga aagaggagaa atactagatg cgtaaaggcg aagagctgtt
     4801 cactggtgtc gtccctattc tggtggaact ggatggtgat gtcaacggtc ataagttttc
     4861 cgtgcgtggc gagggtgaag gtgacgcaac taatggtaaa ctgacgctga agttcatctg
     4921 tactactggt aaactgccgg ttccttggcc gactctggta acgacgctga cttatggtgt
     4981 tcagtgcttt gctcgttatc cggaccatat gaagcagcat gacttcttca agtccgccat
     5041 gccggaaggc tatgtgcagg aacgcacgat ttcctttaag gatgacggca cgtacaaaac
     5101 gcgtgcggaa gtgaaatttg aaggcgatac cctggtaaac cgcattgagc tgaaaggcat
     5161 tgactttaaa gaggacggca atatcctggg ccataagctg gaatacaatt ttaacagcca
     5221 caatgtttac atcaccgccg ataaacaaaa aaatggcatt aaagcgaatt ttaaaattcg
     5281 ccacaacgtg gaggatggca gcgtgcagct ggctgatcac taccagcaaa acactccaat
     5341 cggtgatggt cctgttctgc tgccagacaa tcactatctg agcacgcaaa gcgttctgtc
     5401 taaagatccg aacgagaaac gcgatcatat ggttctgctg gagttcgtaa ccgcagcggg
     5461 catcacgcat ggtatggatg aactgtacaa atgaccaggc atcaaataaa acgaaaggct
     5521 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag
     5581 agtcacactg gctcaccttc gggtgggcct ttctgcgttt ataggtctca gaaatactag
     5641 tactgcag
//
