LOCUS       sequence-455410-        6525 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express node 1467 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 6525)
  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 6525)
  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: pAGK61
FEATURES             Location/Qualifiers
     source          1..6525
                     /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             2780..2800
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2828..3052
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3087..3327
                     /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     3087..3107
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3258..3277
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3656..3870
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     3791..3810
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(4010..4026)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4010..4026)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4023..4045)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4034..4050
                     /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(4058..4064,4065..4082,4083..4088))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4103..4124
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4183..4378
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4229..4364
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4384..4518
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4421..4440)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4475..4494
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4586..4603)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4757..5345)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4837..4856)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5516..6307,6308..6376))
                     /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     6139..6158
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6377..6481)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        join(6512..6525,1..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 gaacgacaac aagaacatcc tcgttaagga gtgggatgcc ttcattagcg aaaacatccg
     1801 cggcgcaaag actcagatcg ctgtcaagac ctacgtgaat aagcttattt ccaataacac
     1861 tgtcgtgatc cttgatttta accacctggc cctgcttctg ggcattgagg tagacgttct
     1921 ggccaggatc acatcacata gcgaaagctt ttattacgac ttcgagatcc ctaagcgtga
     1981 tggcaagtca agaaacatct ccgccccata ccccgtgctc ctgcagtctc agaggtggat
     2041 ttacaagaac attcttagca agcagccaat ccacgattgt agcaagggct tcaagaaaaa
     2101 cacttcaatc gtggacaacg caaaggtgca cctgaaccaa aaaatcctgt tgaagatgga
     2161 catcgaagat ttctttccgt ctatcaagct gaaccgggtg atctccgtgt tcagggtgct
     2221 tggatacacc aaaaagatct cctattacct cgcgtctatc tgctgttaca agggtgctct
     2281 cccccagggc ggtgtgacga gtccaagcct gtccaacatc attactaagc gcctcgacta
     2341 ccgtctgaac ggcctggcat ccaaatttga cctgaagtac acccggtacg cagatgactt
     2401 tactttctcc ggtgagtact tgccgatcaa gattatcgat tatattaaga gtatcgtgag
     2461 caacgagggc ttcaagatca aggacagtaa gactaaaatg atcggtagca aacgccaaaa
     2521 gatcgtgact gggatcagca tctcttccgg taagatgacc attcctagga agacgaagcg
     2581 tgaactgcgt aagaacgtcc actacctgct taaaaatggt ctgtccaagc atctggaaca
     2641 caaccgcatc aacgacccca tctataccga gcgcatgatt ggctacttgc atttttggca
     2701 ctctgtggaa cccaacaata agtacgtaaa agaaagtctc aagaaactga aggactactc
     2761 catccaactg gactctccac caaagaagaa aagaaaggtc tgatcccggt tagtaatgag
     2821 tttaaacctg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     2881 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     2941 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     3001 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggagatctgc
     3061 gaaatcggat ccagctgaat ctaagtgagg gcctatttcc catgattcct tcatatttgc
     3121 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     3181 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     3241 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     3301 tggctttata tatcttgtgg aaaggacgaa acgtagagga tggggaatga aactagcacg
     3361 ttggaggtgt attcgcctct tagattaacg ctggcgttaa ccatactagt gccttaaaag
     3421 gcactagtat agttccacaa acggcagaag ctggaggagg aagggcctga gtccgagcag
     3481 aagaacaagt tggccaaagt tctcccatca catcaaccgg tggcgcattg ccacgaagca
     3541 ggccaatggg gaggacatcg atgtcaggaa ctatactact aaatctactt cgtagtattt
     3601 agcagttaga ataaattaca atgaaagtag ttttcccatc ctctacgttt tttttgaacg
     3661 ctgacgtcat caacccgctc caaggaatcg cgggcccagt gtcactaggc gggaacaccc
     3721 agcgcgcgtg cgccctggca ggaagatggc tgtgagggac aggggagtgg cgccctgcaa
     3781 tatttgcatg tcgctatgtg ttctgggaaa tcaccataaa cgtgaaatgt ctttggattt
     3841 gggaatctta taagttctgt atgagaccac gaaagagtcc gagcagaaga agaagtttca
     3901 gagctatgct ggaaacagca tagcaagttg aaataaggct agtccgttat caacttgaaa
     3961 aagtggcacc gagtcggtgc tttttttttt ttagcttggc gtaatcatgg tcatagctgt
     4021 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4081 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4141 tgcccgcttt ccagtcggga aacctgtcgt gccagcggat ccgcatctca attagtcagc
     4201 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     4261 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     4321 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     4381 gctaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4441 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4501 tcttatcatg tctggatccg ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4561 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4621 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4681 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4741 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4801 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4861 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4921 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4981 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5041 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5101 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5161 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5221 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5281 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5341 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5401 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5461 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5521 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5581 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5641 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5701 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5761 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5821 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5881 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5941 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6001 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6061 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6121 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6181 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6241 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6301 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6361 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6421 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6481 gcacatttcc ccgaaaagtg ccacctgggt cgacattgat tattg
//
