LOCUS       sequence-455407-        6378 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Express node 482 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 6378)
  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 6378)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pAGK57
FEATURES             Location/Qualifiers
     source          1..6378
                     /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             2687..2707
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2735..2959
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2994..3234
                     /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     2994..3014
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3165..3184
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3509..3723
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     3644..3663
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     primer_bind     complement(3863..3879)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3863..3879)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3876..3898)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3887..3903
                     /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(3911..3917,3918..3935,3936..3941))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3956..3977
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4036..4231
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4082..4217
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4237..4371
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4274..4293)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4328..4347
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4439..4456)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4610..5198)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4690..4709)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5369..6160,6161..6229))
                     /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     5992..6011
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6230..6334)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        join(6365..6378,1..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 gctggatgct gacattatca agatttccga ggctttggat gtccccgtga acaagctcta
     1801 catgctgagc aataactaca agcctcgtaa aaagcgccgt ggcagcacat acgaaaacta
     1861 ccataccgtt gtaatccatc acggacgtgg tcacaaaaac cgtaggctca gcgttcctaa
     1921 cgcgtttctg aagatggtgc agagacgcat tctggagtgc tatttgtatc agctcgaggt
     1981 gagcgaccac tctacggcct actgcaaggg gaagagcctg ctcgataatg ccagtcccca
     2041 cgtgggtaag gaatgcgttt tgaagctgga catctcccat tttttcgaca gcatcgacga
     2101 tgacatggtt tatctggtga tgaaacatct tgggctgtct gtcccagcaa cagctctcct
     2161 gacccacctg tgcacttaca aaacccgcct gccgcagggg gcacctacaa gcccctacat
     2221 cgccaacctg gtgatgaagc agttcgacga aaagctgggc tgttggtgcc gccggaacaa
     2281 tatcacctac actcgctact gcgatgacat gactttctcc ggcacaaaat ccgcgattag
     2341 agagagtaga attatctcca aggtgcgcag taaactgtac cacatgggtt tcaccctcaa
     2401 cgacaaaaag acagtgttta tctcttccag tcagcaacag agagttacag gtattgtagt
     2461 gaacgagaaa cccgtactga gccgctctca acgcagggct atccgccaag aggtatatta
     2521 ctgccagaag tacggcgctg tcgagtctat caatcgtaag ggtctcgaca tcccttccga
     2581 taagtatctg cgttcacttc tcggccagat cgcatttgct atgcagatcg gccccgataa
     2641 cgccgagatg cggcgttact tcgataccat caagaacctc atcccaccaa agaagaaaag
     2701 aaaggtctga tcccggttag taatgagttt aaacctgtgc cttctagttg ccagccatct
     2761 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     2821 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     2881 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     2941 gatgcggtgg gctctatgga gatctgcgaa atcggatcca gctgaatcta agtgagggcc
     3001 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg
     3061 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     3121 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     3181 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaacc
     3241 gtaaaacagc cgtcggcagc gaatttgcac aagcaaagct gcggcgagtc gccgcggatg
     3301 atttcaggaa tattaccgat ataaaaagtt cacaaacggc agaagctgga ggaggaaggg
     3361 cctgagtccg agcagaagaa cctgttcgga aaagttctcc catcacatca accggtggcg
     3421 cattgccacg aagcaggcca atggggagga catcgatgtc agaacatttt aatgcttttg
     3481 tgccgtcggc tgttttacgg ttttttttga acgctgacgt catcaacccg ctccaaggaa
     3541 tcgcgggccc agtgtcacta ggcgggaaca cccagcgcgc gtgcgccctg gcaggaagat
     3601 ggctgtgagg gacaggggag tggcgccctg caatatttgc atgtcgctat gtgttctggg
     3661 aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc ttataagttc tgtatgagac
     3721 cacgaaagag tccgagcaga agaagaagtt tcagagctat gctggaaaca gcatagcaag
     3781 ttgaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
     3841 tttttagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     3901 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     3961 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4021 cgtgccagcg gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     4081 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4141 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     4201 aggctttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat
     4261 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4321 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgctgcatt
     4381 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4441 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4501 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4561 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4621 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4681 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4741 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4801 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4861 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4921 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4981 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5041 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5101 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5161 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5221 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5281 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5341 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5401 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5461 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5521 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5581 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5641 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5701 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5761 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5821 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5881 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5941 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6001 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6061 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6121 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6181 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6241 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6301 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6361 ggtcgacatt gattattg
//
