LOCUS       sequence-351770-        2367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ACTB N-term PBS 13 RT 29 Bxb1 AttB 46 atgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2367)
  AUTHORS   Yarnall MTN, Ioannidi EI, Schmitt-Ulms C, Krajeski RN, Lim J, 
            Villiger L, Zhou W, Jiang K, Garushyants SK, Roberts N, Zhang L, 
            Vakulskas CA, Walker JA 2nd, Kadina AP, Zepeda AE, Holden K, Ma H, 
            Xie J, Gao G, Foquet L, Bial G, Donnelly SK, Miyata Y, Radiloff DR, 
            Henderson JM, Ujita A, Abudayyeh OO, Gootenberg JS
  TITLE     Drag-and-drop genome insertion of large sequences without 
            double-strand DNA cleavage using CRISPR-directed integrases.
  JOURNAL   Nat Biotechnol. 2022 Nov 24. doi: 10.1038/s41587-022-01527-4.
  PUBMED    36424489
REFERENCE   2  (bases 1 to 2367)
  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. 2022 Nov 24. doi: 10.1038/s41587-022-01527-4."
COMMENT     Sequence Label: pDY0207 ACTB atgRNA with v1 scaffold
FEATURES             Location/Qualifiers
     source          1..2367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(152..256)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        363..611
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     363..383
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     534..553
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        632..707
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    741..778
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     rep_origin      complement(899..1487)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(979..998)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1658..2367,1..82,83..151))
                     /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     2281..2300
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
       61 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
      121 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
      181 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
      241 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgct agctgtacaa
      301 aaaagcaggc tttaaaggaa ccaattcagt cgactggatc cggtaccaag gtcgggcagg
      361 aagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
      421 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
      481 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
      541 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
      601 gacgaaacac cgctattctc gcagctcacc agttttagag ctagaaatag caagttaaaa
      661 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcgac gagcgcggcg
      721 atatcatcat ccatggccgg atgatcctga cgacggagac cgccgtcgtc gacaagccgg
      781 cctgagctgc gagaattttt ttaagcttgg gccgctcgag gtacctctct acatatgaca
      841 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
      901 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
      961 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     1021 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     1081 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     1141 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     1201 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     1261 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     1321 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     1381 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     1441 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     1501 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     1561 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     1621 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     1681 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     1741 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     1801 atccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     1861 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     1921 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     1981 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     2041 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     2101 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     2161 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     2221 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     2281 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     2341 ggcgaaaact ctcaaggatc ttaccgc
//
