LOCUS       sequence-435415-        9212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pAGD248 derivative with the retron Eco1 msr, sfGFP flanked by 2 BsaI
            sites , and the rest of the retron recombineering operon (Eco1 RT, 
            CspRect, MutL-E32K) downstream of the pBad promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9212)
  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 9212)
  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: pAGD335
FEATURES             Location/Qualifiers
     source          1..9212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(11..889)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        916..1200
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1123..1142
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1227..1235
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     RBS             1532..1543
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1550..2263
                     /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      2275..2346
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2362..2389
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             3449..3457
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     terminator      6176..6270
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6379..7329)
                     /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"
     rep_origin      7377..7599
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(8221..9012,9013..9081))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8844..8863
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9082..9186)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catcgattta ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac
       61 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaag tagagttgat
      121 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca
      181 gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct
      241 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga
      301 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat
      361 tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct
      421 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga
      481 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg
      541 tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt
      601 aaacccactg gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc
      661 ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca
      721 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt
      781 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg
      841 cattaaacga gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac
      901 tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg
      961 tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt
     1021 aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca
     1081 gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat
     1141 ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat
     1201 acccgttttt ttgggaattc gagctctaag gaggttataa aaaagattcc gtatgcgcac
     1261 ccttagcgag aggtttatca ttaaggtcaa cctctggatg ttgtttcggc atcctgcatt
     1321 gaatctgagt tactgtctgt tttcctgaat tctgttggga attcttctag aggtagagag
     1381 accgaaagtg aaacgtgatt tcatgcgtca ttttgaacat tttgtaaatc ttatttaata
     1441 atgtgtgcgg caattcacat ttaatttatg aatgttttct taacatcgcg gcaactcaag
     1501 aaacggcagg ttcggatctt agctactaga gaaagaggag aaatactaga tgcgtaaagg
     1561 cgaagagctg ttcactggtg tcgtccctat tctggtggaa ctggatggtg atgtcaacgg
     1621 tcataagttt tccgtgcgtg gcgagggtga aggtgacgca actaatggta aactgacgct
     1681 gaagttcatc tgtactactg gtaaactgcc ggttccttgg ccgactctgg taacgacgct
     1741 gacttatggt gttcagtgct ttgctcgtta tccggaccat atgaagcagc atgacttctt
     1801 caagtccgcc atgccggaag gctatgtgca ggaacgcacg atttccttta aggatgacgg
     1861 cacgtacaaa acgcgtgcgg aagtgaaatt tgaaggcgat accctggtaa accgcattga
     1921 gctgaaaggc attgacttta aagaggacgg caatatcctg ggccataagc tggaatacaa
     1981 ttttaacagc cacaatgttt acatcaccgc cgataaacaa aaaaatggca ttaaagcgaa
     2041 ttttaaaatt cgccacaacg tggaggatgg cagcgtgcag ctggctgatc actaccagca
     2101 aaacactcca atcggtgatg gtcctgttct gctgccagac aatcactatc tgagcacgca
     2161 aagcgttctg tctaaagatc cgaacgagaa acgcgatcat atggttctgc tggagttcgt
     2221 aaccgcagcg ggcatcacgc atggtatgga tgaactgtac aaatgaccag gcatcaaata
     2281 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     2341 gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttataggtct
     2401 cagaaatact agtactgcag gcggcaaata attttgttta ctttaagaag gagatataca
     2461 tatgaaatcc gctgaatatt tgaacacttt tagattgaga aatctcggcc tacctgtcat
     2521 gaacaatttg catgacatgt ctaaggcgac tcgcatatct gttgaaacac ttcggttgtt
     2581 aatctataca gctgattttc gctataggat ctacactgta gaaaagaaag gcccagagaa
     2641 gagaatgaga accatttacc aaccttctcg agaacttaaa gccttacaag gatgggttct
     2701 acgtaacatt ttagataaac tgtcgtcatc tcctttttct attggatttg aaaagcacca
     2761 atctattttg aataatgcta ccccgcatat tggggcaaac tttatactga atattgattt
     2821 ggaggatttt ttcccaagtt taactgctaa caaagttttt ggagtgttcc attctcttgg
     2881 ttataatcga ctaatatctt cagttttgac aaaaatatgt tgttataaaa atctgctacc
     2941 acaaggtgct ccatcatcac ctaaattagc taatctaata tgttctaaac ttgattatcg
     3001 tattcagggt tatgcaggta gtcggggctt gatatatacg agatatgccg atgatctcac
     3061 cttatctgca cagtctatga aaaaggttgt taaagcacgt gattttttat tttctataat
     3121 cccaagtgaa ggattggtta ttaactcaaa aaaaacttgt attagtgggc ctcgtagtca
     3181 gaggaaagtt acaggtttag ttatttcaca agagaaagtt gggataggta gagaaaaata
     3241 taaagaaatt agagcaaaga tacatcatat attttgcggt aagtcttctg agatagaaca
     3301 cgttagggga tggttgtcat ttattttaag tgtggattca aaaagccata ggagattaat
     3361 aacttatatt agcaaattag aaaaaaaata tggaaagaac cctttaaata aagcgaagac
     3421 ctaagatcga aataattttg tttaacttta aggaggtttg gaatgaacca aatcgtgaag
     3481 ttcactgacg actctggcct ggcggttcaa gttactccag acgatgttcg ccgttatatc
     3541 tgtgagaacg ctactgaaaa agaggtgggc ctctttctgc aactctgtca gactcaacgt
     3601 ctgaatccgt ttgtgaaaga cgcttacctg gtgaaatacg gcggtgctcc agcttctatg
     3661 attacttcct atcaagtttt taaccgtcgc gcgtgtcgtg atgctaacta tgatggtatc
     3721 aaatctggtg tggttgttct gcgtgacggt gatgttgtgc ataaacgtgg tgctgcgtgc
     3781 tacaaaaagg cgggtgagga gctcatcggt ggttgggcgg aagttcgctt taaggatggc
     3841 cgcgagactg cgtatgctga ggtggcgctc gacgactatt ccaccggcaa atctaattgg
     3901 gcgaaaatgc cgggtgttat gatcgaaaaa tgcgcgaagg ctgctgcttg gcgcctcgcg
     3961 ttcccggaca cttttcaggg catgtacgct gcggaggaaa tggatcaagc gcaacagcca
     4021 gaacaggtgc gcgctcaggc ggagcaacca gtggatctcc agccaatccg cgaactcttc
     4081 aagccatatt gcgaacactt cggcatcact ccggctgagg gtatgactgc tgtttgtggt
     4141 gcggtgggcg ctgaaggcat gcactctatg accgagcagc aagctcgccg tgctcgcgct
     4201 tggatggagg aagaaatggc tgcgccagct gtggaagcgg agtatgaggt tgttgacgag
     4261 ggcgaggtgt tttaaggatc caggagaata cacatgccaa ttcaggtctt accgccacaa
     4321 ctggcgaacc agattgccgc aggtgaggtg gtcgagcgac ctgcgtcggt agtcaaagaa
     4381 ctagtgaaaa acagcctcga tgcaggtgcg acgcgtatcg atattgatat cgaacgcggt
     4441 ggggcgaaac ttatccgcat tcgtgataac ggctgcggta tcaaaaaaga tgagctggcg
     4501 ctggcgctgg ctcgtcatgc caccagtaaa atcgcctctc tggacgatct cgaagccatt
     4561 atcagcctgg gctttcgcgg tgaggcgctg gcgagtatca gttcggtttc ccgcctgacg
     4621 ctcacttcac gcaccgcaga acagcaggaa gcctggcagg cctatgccga agggcgcgat
     4681 atgaacgtga cggtaaaacc ggcggcgcat cctgtgggga cgacgctgga ggtgctggat
     4741 ctgttctaca acaccccggc gcggcgcaaa ttcctgcgca ccgagaaaac cgaatttaac
     4801 cacattgatg agatcatccg ccgcattgcg ctggcgcgtt tcgacgtcac gatcaacctg
     4861 tcgcataacg gtaaaattgt gcgtcagtac cgcgcagtgc cggaaggcgg gcaaaaagaa
     4921 cggcgcttag gcgcgatttg cggcaccgct tttcttgaac aagcgctggc gattgaatgg
     4981 caacacggcg atctcacgct acgcggctgg gtggccgatc caaatcacac cacgcccgca
     5041 ctggcagaaa ttcagtattg ctacgtgaac ggtcgcatga tgcgcgatcg cctgatcaat
     5101 cacgcgatcc gccaggcctg cgaagacaaa ctgggggccg atcagcaacc ggcatttgtg
     5161 ttgtatctgg agatcgaccc acatcaggtg gacgtcaacg tgcaccccgc caaacacgaa
     5221 gtgcgtttcc atcagtcgcg tctggtgcat gattttatct atcagggcgt gctgagcgtg
     5281 ctacaacagc aactggaaac gccgctaccg ctggacgatg aaccccaacc tgcaccgcgt
     5341 tccattccgg aaaaccgcgt ggcggcgggg cgcaatcact ttgcagaacc ggcagctcgt
     5401 gagccggtag ctccgcgcta cactcctgcg ccagcatcag gcagtcgtcc ggctgccccc
     5461 tggccgaatg cgcagccagg ctaccagaaa cagcaaggtg aagtgtatcg ccagcttttg
     5521 caaacgcccg cgccgatgca aaaattaaaa gcgccggaac cgcaggaacc tgcacttgcg
     5581 gcgaacagtc agagttttgg tcgggtactg actatcgtcc attccgactg tgcgttgctg
     5641 gagcgcgacg gcaacatttc acttttatcc ttgccagtgg cagaacgttg gctgcgtcag
     5701 gcacaattga cgccgggtga agcgcccgtt tgcgcccagc cgctgctgat tccgttgcgg
     5761 ctaaaagttt ctgccgaaga aaaatcggca ttagaaaaag cgcagtctgc cctggcggaa
     5821 ttgggtattg atttccagtc agatgcacag catgtgacca tcagggcagt gcctttaccc
     5881 ttacgccaac aaaatttaca aatcttgatt cctgaactga taggctacct ggcgaagcag
     5941 tccgtattcg aacctggcaa tattgcgcag tggattgcac gaaatctgat gagcgaacat
     6001 gcgcagtggt caatggcaca ggccataacc ctgctggcgg acgtggaacg gttatgtccg
     6061 caacttgtga aaacgccgcc gggtggtctg ttacaatctg ttgatttaca tccggcgata
     6121 aaagccctga aagatgagtg agtcgtgact gggaaaaccc tggcgactag tcttggactc
     6181 ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt
     6241 tgccgccggg cgttttttat tggtgagaat tccatgggta tggacagttt tccctttgat
     6301 atgtaacggt gaacagttgt tctacttttg tttgttagtc ttgatgcttc actgatagat
     6361 acaagagcca taagaacctc agatccttcc gtatttagcc agtatgttct ctagtgtggt
     6421 tcgttgtttt tgcgtgagcc atgagaacga accattgaga tcatacttac tttgcatgtc
     6481 actcaaaaat tttgcctcaa aactggtgag ctgaattttt gcagttaaag catcgtgtag
     6541 tgtttttctt agtccgttat gtaggtagga atctgatgta atggttgttg gtattttgtc
     6601 accattcatt tttatctggt tgttctcaag ttcggttacg agatccattt gtctatctag
     6661 ttcaacttgg aaaatcaacg tatcagtcgg gcggcctcgc ttatcaacca ccaatttcat
     6721 attgctgtaa gtgtttaaat ctttacttat tggtttcaaa acccattggt taagcctttt
     6781 aaactcatgg tagttatttt caagcattaa catgaactta aattcatcaa ggctaatctc
     6841 tatatttgcc ttgtgagttt tcttttgtgt tagttctttt aataaccact cataaatcct
     6901 catagagtat ttgttttcaa aagacttaac atgttccaga ttatatttta tgaatttttt
     6961 taactggaaa agataaggca atatctcttc actaaaaact aattctaatt tttcgcttga
     7021 gaacttggca tagtttgtcc actggaaaat ctcaaagcct ttaaccaaag gattcctgat
     7081 ttccacagtt ctcgtcatca gctctctggt tgctttagct aatacaccat aagcattttc
     7141 cctactgatg ttcatcatct gaacgtattg gttataagtg aacgataccg tccgttcttt
     7201 ccttgtaggg ttttcaatcg tggggttgag tagtgccaca cagcataaaa ttagcttggt
     7261 ttcatgctcc gttaagtcat agcgactaat cgctagttca tttgctttga aaacaactaa
     7321 ttcagacata catctcaatt ggtctaggtg attttaatca ctataccaat tgagatgggc
     7381 tagtcaatga taattactag tccttttcct ttgagttgtg ggtatctgta aattctgcta
     7441 gacctttgct ggaaaacttg taaattctgc tagaccctct gtaaattccg ctagaccttt
     7501 gtgtgttttt tttgtttata ttcaagtggt tataatttat agaataaaga aagaataaaa
     7561 aaagataaaa agaatagatc ccagccctgt gtataactca ctactttagt cagttccgca
     7621 gtattacaaa aggatgtcgc aaacgctgtt tgctcctcta caaaacagac cttaaaaccc
     7681 taaaggctta agtagcaccc tcgcaagctc ggttgcggcc gcaatcgggc aaatcgctga
     7741 atattccttt tgtctccgac catcaggcac ctgagtcgct gtctttttcg tgacattcag
     7801 ttcgctgcgc tcacggctct ggcagtgaat gggggtaaat ggcactacag gcgcctttta
     7861 tggattcatg caaggaaact acccataata caagaaaagc ccgtcacggg cttctcaggg
     7921 cgttttatgg cgggtctgct atgtggtgct atctgacttt ttgctgttca gcagttcctg
     7981 ccctctgatt ttccagtctg accacttcgg attatcccgt gacaggtcat tcagactggc
     8041 taatgcaccc agtaaggcag cggtatcatc aacggggtct gacgctcagt ggaacgaaaa
     8101 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     8161 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     8221 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     8281 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     8341 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     8401 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     8461 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     8521 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     8581 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     8641 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     8701 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     8761 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     8821 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     8881 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     8941 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     9001 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     9061 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     9121 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     9181 tccgcgcaca tttccccgaa aagtgccacc tg
//
