LOCUS       sequence-434395-        4294 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of type III-A crRNA from Streptococcus 
            thermophilus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4294)
  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 4294)
  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: pAN099
FEATURES             Location/Qualifiers
     source          1..4294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      153..741
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     642..661
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        795..998
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      1029..1038
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1038..1061
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1068..1088
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1827..1874
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        1905..2108
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             2145..2744
                     /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(2145..2164)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2641..2661
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2745..2792
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        2823..3071
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2823..2843
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2994..3013
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3225..3243)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3311..3415
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3416..3484,3485..4276)
                     /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(3634..3653)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
       61 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
      121 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
      181 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
      241 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
      301 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
      361 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
      421 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
      481 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
      541 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
      601 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
      661 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
      721 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttcttaag gccggccgga
      781 gaataaaagt ctaagtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      841 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      901 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      961 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatct
     1021 ctagagccgc caccatggat tacaaagacg atgacgataa gatggcgcct aagaagaaac
     1081 gcaaagtgcg gggcatgaaa aagctcgtgt tcacctttaa gcggatcgac caccctgctc
     1141 aggacctggc cgtgaaattc cacggcttcc tgatggaaca gctggatagc gactacgtgg
     1201 actacctgca ccagcagcag accaacccct acgccacaaa ggtgatccag ggcaaagaga
     1261 acacccagtg ggtcgtgcat ctgctgacag acgacatcga ggacaaggtg ttcatgaccc
     1321 tgctgcagat caaggaagtg tccctgaacg acctgcctaa gttgtctgtg gaaaaggtgg
     1381 aaatccagga gctgggcgct gataagctgc tcgagatctt caacagcgag gaaaaccaga
     1441 cctacttcag catcatcttc gagacaccta caggctttaa aagccagggc agctacgtga
     1501 tcttccccag catgcggctg atctttcaga gcctgatgca gaagtacggc agactggtgg
     1561 aaaaccagcc tgagatcgag gaagataccc tggactacct gagcgagcac agcaccatca
     1621 ccaattacag actggaaaca agctacttca gagtgcatag acagagaatc cccgccttcc
     1681 ggggcaagct gaccttcaag gtgcagggag cccagacact gaaggcctac gtgaagatgc
     1741 tgctgacctt cggcgagtac agcggcctgg gcatgaaaac cagcctggga atgggcggca
     1801 tcaagctgga agaaagaaag gactgacggc aataaaaaga cagaataaaa cgcacggtgt
     1861 tgggtcgttt gttcgtgcga tagagggatc ccgcattgaa ttatgtgatg cggttttggc
     1921 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     1981 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     2041 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     2101 gcagagctcg tttagtgaac cgtcagatct ctagagccgc caccatgacc gagtacaagc
     2161 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     2221 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     2281 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     2341 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     2401 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     2461 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     2521 ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     2581 ccggagtgga ggcggcggag cgcgccgggg tgcccgcctt cctggaaacc tccgcgcccc
     2641 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     2701 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgacggcaa taaaaagaca
     2761 gaataaaacg cacggtgttg ggtcgtttgt tcgagcagat tgtactgaga gtgcaccggt
     2821 tggagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     2881 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     2941 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     3001 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     3061 gacgaaacac cgatataaac ctaattacct cgagagggga cggaaacccg tcttcgatga
     3121 agcgattcag aagacttgat ataaacctaa ttacctcgag aggggacttt tttacatgtg
     3181 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     3241 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     3301 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3361 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3421 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3481 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3541 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3601 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     3661 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3721 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3781 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     3841 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     3901 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     3961 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4021 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4081 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4141 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4201 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4261 gattaagcat tggtaactgt cagaccaagt ttac
//
