LOCUS       sequence-467885-        3353 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PseCAST crRNA targeting AAVS1-5, with 1 kb donor transposon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3353)
  AUTHORS   Witte IP, Lampe GD, Eitzinger S, Miller SM, Berrios KN, McElroy AN, 
            King RT, Stringham OG, Gelsinger DR, Vo PLH, Chen AT, Tolar J, 
            Osborn MJ, Sternberg SH, Liu DR
  TITLE     Programmable gene insertion in human cells with a laboratory-evolved
            CRISPR-associated transposase.
  JOURNAL   Science. 2025 May 15;388(6748):eadt5199. doi: 
            10.1126/science.adt5199. Epub 2025 May 15.
  PUBMED    40373119
REFERENCE   2  (bases 1 to 3353)
  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: "Science."; 
            date: "2025-05-15"; volume: "388(6748):eadt5199. doi"; pages: " 
            10.1126/science.adt5199. Epub 2025 May 15"
COMMENT     Sequence Label: pU6-AAVS1-5-1kbdonor
FEATURES             Location/Qualifiers
     source          1..3353
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1243..1491
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1243..1263
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1414..1433
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    1497..1511
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    1557..1571
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     primer_bind     complement(1613..1631)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1627..1674
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      complement(2765..3353)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2845..2864)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgct agctgtacaa aaaagcaggc tttaaaggaa
     1201 ccaattcagt cgactggatc cggtaccaag gtcgggcagg aagagggcct atttcccatg
     1261 attccttcat atttgcatat acgatacaag gctgttagag agataattag aattaatttg
     1321 actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg
     1381 tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga
     1441 aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac cgtgacctgc
     1501 cgtataggca gctgaaaata atcctgtccc tagtggcccc actgtggggt ggtgacctgc
     1561 cgtataggca gctgaaaatt tttttttttc ctaggctgct gcgctgctgc caccgctgag
     1621 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     1681 cctcaggcat ttgttgtcgc tgcaaccata gtttgacata atgcaaccat accttgacat
     1741 aattaaagtc atactctgac ataaagcccc gggcgtggtg ccgattctgg tggaactgga
     1801 tggtgggaaa agccctggaa gatgccatga catggtcacc accgggtaaa ggggatgaaa
     1861 ttcagcgtgc gcggcgaagg tgaaggcgat gcaaccaatg gtaaactgac gctgaagttt
     1921 atttgcacca cgggtaaact gccggttccg tggcctaccc tggtcaccac gctgacgtat
     1981 ggtgttcagt gtttcagtcg ttacccggat cacatgaaac gccacgactt tttcaagtcc
     2041 gcgatgccgg aaggttatgt ccaagaacgt accatctcat ttaaagatga cggcacctac
     2101 aaaacgcgcg ccgaagtgaa attcgaaggt gatacgctgg ttaaccgtat tgaactgaaa
     2161 ggcatcgatt ttaaggaaga cggtaatatt ctgggccata aactggaata taacttcaat
     2221 tcgcacaacg tgtacatcac cgcagataag cagaagaacg gtatcaaggc taacttcaag
     2281 atccgccata atgtggaaga tggcagcgtt caactggccg accactatca gcaaaacacc
     2341 ccgattggtg atggcccggt cctgctgccg gacaatcatt acctgagcac gcagtctgtg
     2401 ctgagtaaag atccgaacga aaagcgtgac cacatggtcc tgctggaatt cgtgaccgcg
     2461 gccggcatca cgcacggtat ggacgaactg tataaaggct cataacatcc cctttacccg
     2521 gtgggctggt ttttttgttt ttagcgttat aacttttatt tcactatgtc actgtatggt
     2581 tgcattttat gtcagtgaat ggttgcaggt tttttgtaag ttattgatat gcctatccag
     2641 tgtatgtcac gctttgcttg cagcgacaaa gcttgggccg ctcgaggtac ctctctacat
     2701 atgacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     2761 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     2821 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     2881 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2941 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3001 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3061 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3121 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3181 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     3241 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3301 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caa
//
