LOCUS       sequence-455406-        6470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express node 1429 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 6470)
  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 6470)
  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: pAGK59
FEATURES             Location/Qualifiers
     source          1..6470
                     /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             2744..2764
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2792..3016
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3051..3299
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3051..3071
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3222..3241
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3601..3815
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     3736..3755
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(3955..3971)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3955..3971)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3968..3990)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3979..3995
                     /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(4003..4009,4010..4027,4028..4033))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4048..4069
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4128..4323
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4174..4309
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4329..4463
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4366..4385)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4420..4439
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4531..4548)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4702..5290)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4782..4801)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5461..6252,6253..6321))
                     /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     6084..6103
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6322..6426)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        join(6457..6470,1..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 gcgtagggag tgggaggact tttatctgga gaagggcatt aacacaaatt tcattagtac
     1801 ctacctggac tacatcaaga ccctcgagtc ccgtaatatt cctgtcatct ttgaggtcga
     1861 gcatttgtcc aagctgatcg gcatcaagta ccctgaaatc aacgacattg tgttcggaac
     1921 gaagtccttc tacagggaat tcaagatccc taaaaagaaa ggtggctacc gtaagatcag
     1981 ctcaccttac cctagtctgc gcaaatgcca gacttggatc cacgaaaagc ttctgtattc
     2041 tctgcgtact cacaagaatg ccttcgccta caaaaaggga aaatccatca ttgacaacgc
     2101 tgagcagcac ctcaacaatc acttcctgtt gaagatcgac atcaaggact tctttccatc
     2161 catcccaatt aactgggtta tcaagctctt cgactccctg ggttatgcca agaatgtgtc
     2221 ctacgctctt gcctctctgt gttgcctgga tggctgcctt ccgcaaggcg ggattacctc
     2281 tccatccctg tccaatatcc ttctgtaccc cctggacgag cgtctgtcta aactggccct
     2341 caagttcaac ctgaaatatt cccgctacgc cgatgacatg gttttcagcg gggaggctat
     2401 ccctagcaag ttcatctctt atgtgacaga cattatcgaa agctacggct ttattctcaa
     2461 caaggagaag accaagctcc tgaacaatac taagcagaag atcgtcactg gcatcgacgt
     2521 gagcaaggat aaactccaac tgccccgcaa gacccgccgt gaaatccgta aaaccatgca
     2581 ttacattaaa aagtacggca tcttctccca cattgacaac gaaaaaatca aggatcccaa
     2641 ctacatcaaa tcactggagg gaaagatccg gttctggctg caagtcgagc ctgacaacca
     2701 agaggcccgc agctacattg agcacattaa tcagatgaag ccaccaaaga agaaaagaaa
     2761 ggtctgatcc cggttagtaa tgagtttaaa cctgtgcctt ctagttgcca gccatctgtt
     2821 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2881 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2941 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     3001 gcggtgggct ctatggagat ctgcgaaatc ggatccagct gaatctaagt gagggcctat
     3061 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     3121 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     3181 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     3241 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaaagact
     3301 tgtagaaact agcacgttgg agtgtagtcg ctccttggtt aagggtcact ttaaccacga
     3361 ctatacggat aactccgtat agtcgcttcg aatagcgttg ctattctcgc ggttctttac
     3421 aaacggcaga agctggagga ggaagggcct gagtccgagc agaagaaatc tgctaggaaa
     3481 gttctcccat cacatcaacc ggtggcgcat tgccacgaag caggccaatg gggaggacat
     3541 cgatgtcaag agaacttagg tgtcaggtat agaaagtagt ttctacaagt cttttttttt
     3601 gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa
     3661 cacccagcgc gcgtgcgccc tggcaggaag atggctgtga gggacagggg agtggcgccc
     3721 tgcaatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg
     3781 gatttgggaa tcttataagt tctgtatgag accacgaaag agtccgagca gaagaagaag
     3841 tttcagagct atgctggaaa cagcatagca agttgaaata aggctagtcc gttatcaact
     3901 tgaaaaagtg gcaccgagtc ggtgcttttt tttttttagc ttggcgtaat catggtcata
     3961 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4021 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4081 ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgca tctcaattag
     4141 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4201 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4261 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4321 aaaaagctaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4381 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4441 atgtatctta tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg cggggagagg
     4501 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4561 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4621 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4681 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4741 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4801 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4861 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4921 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4981 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5041 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5101 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5161 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5221 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5281 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5341 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5401 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5461 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5521 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5581 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5641 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5701 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5761 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5821 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5881 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5941 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6001 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6061 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6121 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6181 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6241 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6301 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6361 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6421 tccgcgcaca tttccccgaa aagtgccacc tgggtcgaca ttgattattg
//
