LOCUS       sequence-455303-        6366 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express node 2 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 6366)
  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 6366)
  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: pAGK55
FEATURES             Location/Qualifiers
     source          1..6366
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        368..645
                     /label=chicken beta-actin promoter
     intron          646..1662
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1586..1608
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1670..1689
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2672..2692
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2720..2944
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2979..3219
                     /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     2979..2999
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3150..3169
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3497..3711
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     3632..3651
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(3851..3867)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3851..3867)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3864..3886)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3875..3891
                     /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(3899..3905,3906..3923,3924..3929))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3944..3965
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4024..4219
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4070..4205
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4225..4359
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4262..4281)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4316..4335
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4427..4444)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4598..5186)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4678..4697)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5357..6148,6149..6217))
                     /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     5980..5999
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6218..6322)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        join(6353..6366,1..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 gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg
      541 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
      601 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
      661 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
      721 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
      781 cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc
      841 cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg
      901 gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg
      961 gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg
     1021 tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga
     1081 gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt
     1141 gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc
     1201 gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc
     1261 ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg
     1321 gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg
     1381 tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc
     1441 ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg
     1501 ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc
     1561 cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga
     1621 gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg
     1681 gttattgtgc tgtctcatca ttttggcaaa gaattgagct cgatatcggt acgccaccat
     1741 gagcgtgatc aatgggctgg ctggtctcct gaatcagagc gagcttgatg tcgttcttta
     1801 cctccagagt gctagtcgca agtacaaggt gtataagatc ccaaagagga ctaccggctt
     1861 ccggattatc gcccagccag caaaggctct gaaggattac cagcgcgcct tcctgcagtt
     1921 gtataggctg cccgtccaca agtgtgctat ggcataccag aaaggtaagt ctgtgaggga
     1981 taacgccctg gtccacgccc agaactcctt tcttctgaag acagacctgg aggatttttt
     2041 caacagcatc accccagata ttttctggga gagcatcgag cgctccccaa gtgacactcc
     2101 ccagatctct gcggaggaca agggctacgt agagcatctg ctcttttggc aacctgccaa
     2161 gaggagcaag cgcctgatcc tcagcgtggg cgctccgagt tcccccgtgg tcagcaattt
     2221 ctgcctgtat gagttcgaca atctgatcag cgagttctgc ctgcctctga acatcgcata
     2281 cacccgttac gccgatgacc tgacattctc atgtaacacc agggacgtgc tcggggttct
     2341 gcctctgatc attgagaagc tgttgaacaa gctgtaccgt cgcaagctga ggctgaaccg
     2401 cgcgaagacc gtcttctcat ctatggctca taaccgccac gtcaccggtg ttactatcaa
     2461 caatgaggga gaactgagtc ttgggcgtga gcgtaaacgc ttcattaagc acctgattaa
     2521 tcagtaccgg tacgatatga tcgacgaggc cgataaggcg tacctggttg gcctgctcgc
     2581 attcgcgaaa cacatcgagc ctgagttcat cctgcggatg aaccgcaaat acaccactga
     2641 actcatggag cgcatcagac gtcagcagcc accaaagaag aaaagaaagg tctgatcccg
     2701 gttagtaatg agtttaaacc tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2761 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2821 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2881 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     2941 atggagatct gcgaaatcgg atccagctga atctaagtga gggcctattt cccatgattc
     3001 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     3061 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     3121 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     3181 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaaccgtatc aactctttag
     3241 cgtggacgta ttacgtctag tcgggtgatt agccagactc taacttattg aacgcatttg
     3301 gggttgcgaa agtgtcgcaa caaacggcag aagctggagg aggaagggcc tgagtccgag
     3361 cagaagaact cgcagtagaa agttctccca tcacatcaac cggtggcgca ttgccacgaa
     3421 gcaggccaat ggggaggaca tcgatgtcat gcgacacttt cgcttcctcc agtaaagagt
     3481 tgatacggtt ttttttgaac gctgacgtca tcaacccgct ccaaggaatc gcgggcccag
     3541 tgtcactagg cgggaacacc cagcgcgcgt gcgccctggc aggaagatgg ctgtgaggga
     3601 caggggagtg gcgccctgca atatttgcat gtcgctatgt gttctgggaa atcaccataa
     3661 acgtgaaatg tctttggatt tgggaatctt ataagttctg tatgagacca cgaaagagtc
     3721 cgagcagaag aagaagtttc agagctatgc tggaaacagc atagcaagtt gaaataaggc
     3781 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttttt tttagcttgg
     3841 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3901 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     3961 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga
     4021 tccgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4081 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4141 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     4201 aggcctaggc ttttgcaaaa agctaacttg tttattgcag cttataatgg ttacaaataa
     4261 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4321 ttgtccaaac tcatcaatgt atcttatcat gtctggatcc gctgcattaa tgaatcggcc
     4381 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4441 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4501 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4561 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4621 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4681 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4741 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4801 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4861 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4921 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4981 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5041 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5101 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5161 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5221 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5281 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5341 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5401 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5461 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5521 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5581 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5641 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5701 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5761 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5821 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5881 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5941 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6001 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6061 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6121 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6181 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6241 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6301 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctggg tcgacattga
     6361 ttattg
//
