LOCUS       sequence-458565-        6457 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express node 814 retron with split retron ncRNA and gRNA to edit 
            (10bp barcode) EMX1 gene in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6457)
  AUTHORS   Khan AG, Rojas-Montero M, Gonzalez-Delgado A, Lopez SC, Fang RF, 
            Crawford KD, Shipman SL
  TITLE     An experimental census of retrons for DNA production and genome 
            editing.
  JOURNAL   Nat Biotechnol. 2024 Sep 17. doi: 10.1038/s41587-024-02384-z.
  PUBMED    39289529
REFERENCE   2  (bases 1 to 6457)
  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"; journalName: "Nat 
            Biotechnol. 2024 Sep 17. doi: 10.1038/s41587-024-02384-z."
COMMENT     Sequence Label: pAGK58
FEATURES             Location/Qualifiers
     source          1..6457
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        368..647
                     /label=chicken beta-actin promoter
     intron          648..1664
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1588..1610
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1672..1691
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1735..1744
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2746..2766
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2794..3018
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3053..3293
                     /label=U6 promoter
                     /note="Truncated RNA polymerase III promoter for human U6 
                     snRNA used in pSIREN vectors such as 
                     pSIREN-RetroQ-ZsGreen1. Expected transcription start site 
                     is 8bp downstream of this feature."
     primer_bind     3053..3073
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3224..3243
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3588..3802
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     3723..3742
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(3942..3958)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3942..3958)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3955..3977)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3966..3982
                     /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)."
     promoter        complement(join(3990..3996,3997..4014,4015..4020))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4035..4056
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4115..4310
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4161..4296
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4316..4450
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4353..4372)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4407..4426
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4518..4535)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4689..5277)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4769..4788)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5448..6239,6240..6308))
                     /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     6071..6090
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6309..6413)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        join(6444..6457,1..366)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
      421 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
      481 gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc
      541 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
      601 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc
      661 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
      721 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
      781 agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc
      841 tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg
      901 tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg
      961 cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc
     1021 ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt
     1081 gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag
     1141 ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg
     1201 ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg
     1261 ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg
     1321 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt
     1381 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc
     1441 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt
     1501 cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct
     1561 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     1621 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     1681 tggttattgt gctgtctcat cattttggca aagaattgag ctcgatatcg gtacgccacc
     1741 atggagccca atgactacgt aaataggttg cgacatgcta tggaaataag tgaaaacccg
     1801 cgctttagcc ctgagtacat tgctcaatgt tgtacttacg ccgagaatct cctcaagcaa
     1861 gggctgccag ttctgttcga tcaaacgcat attcggaaag ttctggggat ggcggcacct
     1921 cgattgtgtg attatcacag attcacaatc ccaaagcaca acggatctag aattatcacg
     1981 gccccatcta ggaagctgaa gcttcgacaa caatggatat accagaatat ccttatacga
     2041 aaggaggctt caccgtacac gcacggattt gttcctgaac gcagcatcgt gactaacgca
     2101 atcctccata taggatacgc atacacctac tgcgtggata tcacggattt ctttcctagc
     2161 atcactaaga agcaggtctt gcctatattc cgaaatatgg gctatagtgg ttctgctgca
     2221 aatactctct gcgacctctg ttgctatgac ggggtcctcc cccagggggc gcctactagc
     2281 ccatacctca gtaacatgat ttgtcgcgat cttgatgacg aattgggggc tatggcgcgg
     2341 cggttccggg ggatttttac acggtatgcg gatgacatag ctatctccac aaaccaacaa
     2401 cagccgcaac ttttggatgc cttgggactt atcctcggga agcacggatt tcttatgaat
     2461 ctcgataagt gtcgagtcta taatcctgga cagcccaaaa gaattactgg attgaccgtt
     2521 cacaatagag tatcagttcc gaaaaccttt aagcggaaat tgcggcagga aatacattac
     2581 tgtcagaagt tcggagtgac tgcacatttg gaaaacacga aggctgcacg atccatccac
     2641 tatagggaac atctgtatgg aaaggcatac tatgttaaaa tggttgagcc tgagctcggg
     2701 gcgcacttcc tcgatgagtt gtcaaaggta gactggccag agccaccaaa gaagaaaaga
     2761 aaggtctgat cccggttagt aatgagttta aacctgtgcc ttctagttgc cagccatctg
     2821 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2881 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2941 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3001 atgcggtggg ctctatggag atctgcgaaa tcggatccag ctgaatctaa gtgagggcct
     3061 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
     3121 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     3181 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     3241 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaagta
     3301 taaaaccggg aacgatcaga ccggggtgaa ttcgccccct tgatcaaacg gcactaacca
     3361 ctgtttgccg tgcgtgcgta caaacggcag aagctggagg aggaagggcc tgagtccgag
     3421 cagaagaata tggcccataa agttctccca tcacatcaac cggtggcgca ttgccacgaa
     3481 gcaggccaat ggggaggaca tcgatgtcaa cgcacgcacg gcaaacagac agatccatta
     3541 ttattacaat ttatttagtg atcgttcccg gttttatact tttttttgaa cgctgacgtc
     3601 atcaacccgc tccaaggaat cgcgggccca gtgtcactag gcgggaacac ccagcgcgcg
     3661 tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca
     3721 tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct
     3781 tataagttct gtatgagacc acgaaagagt ccgagcagaa gaagaagttt cagagctatg
     3841 ctggaaacag catagcaagt tgaaataagg ctagtccgtt atcaacttga aaaagtggca
     3901 ccgagtcggt gctttttttt ttttagcttg gcgtaatcat ggtcatagct gtttcctgtg
     3961 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4021 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4081 ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca gcaaccatag
     4141 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     4201 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     4261 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctaactt
     4321 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4381 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4441 tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4501 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4561 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4621 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4681 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4741 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4801 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4861 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4921 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4981 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5041 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5101 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5161 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5221 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5281 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5341 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5401 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5461 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5521 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5581 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5641 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5701 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5761 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5821 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5881 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5941 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6001 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6061 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6121 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6181 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6241 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6301 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6361 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6421 ccccgaaaag tgccacctgg gtcgacattg attattg
//
