LOCUS       sequence-437052-        2330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hACTB atgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2330)
  AUTHORS   Fell CW, Schmitt-Ulms C, Tagliaferri DV, Gootenberg JS, Abudayyeh OO
  TITLE     Precise kilobase-scale genomic insertions in mammalian cells using 
            PASTE.
  JOURNAL   Nat Protoc. 2024 Dec 15. doi: 10.1038/s41596-024-01090-z.
  PUBMED    39676077
REFERENCE   2  (bases 1 to 2330)
  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 Protoc.
            2024 Dec 15. doi: 10.1038/s41596-024-01090-z."
COMMENT     Sequence Label: pDY2262 hACTB atgRNA Paired Guide 2
FEATURES             Location/Qualifiers
     source          1..2330
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     393..412
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(631..735)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        842..1090
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     842..862
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1013..1032
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1111..1186
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1187..1224
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     rep_origin      complement(1341..1929)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1421..1440)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2100..2330,1..561,562..630))
                     /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"
ORIGIN
        1 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
       61 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
      121 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
      181 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
      241 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
      301 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
      361 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
      421 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
      481 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
      541 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
      601 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
      661 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
      721 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgcta gctgtacaaa
      781 aaagcaggct ttaaaggaac caattcagtc gactggatcc ggtaccaagg tcgggcagga
      841 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
      901 gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
      961 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     1021 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     1081 acgaaacacc ggaggggaag acggcccggg gttttagagc tagaaatagc aagttaaaat
     1141 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcggct tgtcgacgac
     1201 ggcggtctcc gtcgtcagga tcatgggccg tcttcccttt ttttaagctt gggccgctcg
     1261 aggtacctct ctacatatga catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     1321 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     1381 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     1441 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     1501 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     1561 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     1621 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     1681 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     1741 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     1801 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     1861 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1921 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     1981 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     2041 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     2101 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     2161 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     2221 agtgctgcaa tgataccgcg agatccacgc tcaccggctc cagatttatc agcaataaac
     2281 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
//
