LOCUS       sequence-434397-        3188 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of type III-A crRNA and Cas6 crRNA-processing 
            ribonuclease from Streptococcus thermophilus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3188)
  AUTHORS   Nemudraia A, Nemudryi A, Wiedenheft B
  TITLE     Repair of CRISPR-guided RNA breaks enables site-specific RNA 
            excision in human cells.
  JOURNAL   Science. 2024 Apr 25:eadk5518. doi: 10.1126/science.adk5518.
  PUBMED    38662916
REFERENCE   2  (bases 1 to 3188)
  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. 
            2024 Apr 25:eadk5518. doi: 10.1126/science.adk5518."
COMMENT     Sequence Label: pAN102
FEATURES             Location/Qualifiers
     source          1..3188
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        91..294
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     245..265
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     291..315
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             331..930
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(331..350)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     827..847
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    931..978
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        1009..1257
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1009..1029
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1180..1199
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1411..1429)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1497..1601
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1602..1670,1671..2462)
                     /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     complement(1820..1839)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(2633..3188,1..33)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3122..3141
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttctta
       61 aggcgataga gggatcccgc attgaattat gtgatgcggt tttggcagta catcaatggg
      121 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      181 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      241 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
      301 gtgaaccgtc agatctctag agccgccacc atgaccgagt acaagcccac ggtgcgcctc
      361 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
      421 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
      481 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
      541 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
      601 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
      661 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc
      721 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
      781 gcggagcgcg ccggggtgcc cgccttcctg gaaacctccg cgccccgcaa cctccccttc
      841 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
      901 tggtgcatga cccgcaagcc cggtgcctga cggcaataaa aagacagaat aaaacgcacg
      961 gtgttgggtc gtttgttcga gcagattgta ctgagagtgc accggttgga gggcctattt
     1021 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt
     1081 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     1141 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     1201 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgat
     1261 ataaacctaa ttacctcgag aggggacgga aacccgtctt cgatgaagcg attcagaaga
     1321 cttgatataa acctaattac ctcgagaggg gactttttta catgtgtcag aggttttcac
     1381 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     1441 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     1501 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1561 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1621 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1681 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1741 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1801 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     1861 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1921 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     1981 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2041 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2101 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     2161 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2221 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2281 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2341 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2401 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2461 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2521 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2581 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2641 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2701 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2761 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2821 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2881 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2941 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3001 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3061 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3121 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3181 gatttttg
//
