LOCUS       sequence-455403-        6614 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express node 1531 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 6614)
  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 6614)
  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: pAGK62
FEATURES             Location/Qualifiers
     source          1..6614
                     /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             2894..2914
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2942..3166
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3201..3441
                     /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     3201..3221
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3372..3391
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3745..3959
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     3880..3899
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(4099..4115)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4099..4115)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4112..4134)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4123..4139
                     /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(4147..4153,4154..4171,4172..4177))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4192..4213
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4272..4467
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4318..4453
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4473..4607
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4510..4529)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4564..4583
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4675..4692)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4846..5434)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4926..4945)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5605..6396,6397..6465))
                     /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     6228..6247
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6466..6570)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        join(6601..6614,1..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 gaaaaagtat agcaacaaga cggtgatcga gagtctggaa ctgccaatgc tgacatgctt
     1801 cgacgatctc gtgaaggaac tctctctgag cgttaagctg gtgtactggc tgactaagca
     1861 gggagctgag ggccggtaca agaccttctt tattggcaaa aaggatggtg ataagaggaa
     1921 gatcaatgct ccgagcctgg ctctgaagat cgtacagcgg tgggttctgg agaacatctt
     1981 gtacaagatc aaaacttccc aatattctta cggcttcacc aaggggaagc ggaagggtag
     2041 cccgctggtg tactgcgcag agaagcacaa aaataacctc tatgtcctga agctggatct
     2101 gaagaatttt taccccagta tcaagcgtga aaaggtctat tacgcgtttc tgaacatcgg
     2161 atataacgca gacgtgtcta acatgttgac taacatttgt gtggtcgacg atgagctgcc
     2221 acagggcgcc gttacctccc cctaccttgc aaatttgatc tgccgtaagc tggacctgcg
     2281 catcgcaggc tattgtaaca agcgcaatat cgtctacaca cgctatgctg acgatttggc
     2341 tttctcctgt gatgaccgcg agctcctgcg caagatctac ggcatggtga agaaaatcgt
     2401 cgaagatgaa ggattctgtc tgaaccctaa gaaaactgtc ttcatgacac ccaagaacca
     2461 caaggaagta cttggtgtca ccatcaatga ttctctcctg aaagcaccca aagaaatcaa
     2521 gaggtctatc cgcgcgatga tccactatga ggtggctaca ggcgactaca cgatgaacga
     2581 caaaatccgt ggctatatcg cctacgttga ctccatcgaa aagaattaca agaacaagtg
     2641 catctcctac ctgcaaaaat tgtccgagtc tactctgtgt ctcttccctg atatcgtcaa
     2701 ggcttataac ggcaataaga tctacaagga gcttccagac atgaaggaaa agagggccac
     2761 cgacttcgtg gagttggaag aggccgacga tttttcaaat atgatttatt acgagcatga
     2821 agagtacctg attagcaagg gattgctgga caaaaaggag gacgtgactg acctcgatga
     2881 gccattcttg ccaccaaaga agaaaagaaa ggtctgatcc cggttagtaa tgagtttaaa
     2941 cctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     3001 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     3061 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     3121 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggagat ctgcgaaatc
     3181 ggatccagct gaatctaagt gagggcctat ttcccatgat tccttcatat ttgcatatac
     3241 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     3301 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     3361 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     3421 tatatatctt gtggaaagga cgaaaattag tactgctttc ttagaacgtt acagatacat
     3481 atctgtttac ggaagatggt tctgtcttcc taagaaaatt aacaaacaca gagttactcc
     3541 aatcaatgga gtaaacaaac ggcagaagct ggaggaggaa gggcctgagt ccgagcagaa
     3601 gaaccggcat tacaaagttc tcccatcaca tcaaccggtg gcgcattgcc acgaagcagg
     3661 ccaatgggga ggacatcgat gtcattactc cattgattgg agacaaagag atttaaggta
     3721 agaaagcggt gctaattttt ttttgaacgc tgacgtcatc aacccgctcc aaggaatcgc
     3781 gggcccagtg tcactaggcg ggaacaccca gcgcgcgtgc gccctggcag gaagatggct
     3841 gtgagggaca ggggagtggc gccctgcaat atttgcatgt cgctatgtgt tctgggaaat
     3901 caccataaac gtgaaatgtc tttggatttg ggaatcttat aagttctgta tgagaccacg
     3961 aaagagtccg agcagaagaa gaagtttcag agctatgctg gaaacagcat agcaagttga
     4021 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttttttt
     4081 tagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4141 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4201 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4261 ccagcggatc cgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     4321 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     4381 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     4441 ttttttggag gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct tataatggtt
     4501 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4561 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatccgc tgcattaatg
     4621 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4681 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4741 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4801 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4861 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4921 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4981 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5041 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5101 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5161 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5221 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5281 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5341 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5401 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5461 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5521 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5581 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5641 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5701 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5761 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5821 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5881 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5941 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6001 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6061 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6121 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6181 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6241 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6301 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6361 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6421 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6481 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6541 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgggtc
     6601 gacattgatt attg
//
