LOCUS       sequence-478692-        5162 bp    DNA     circular SYN 15-JUL-2025
DEFINITION  crRNA array expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5162)
  AUTHORS   Yang LZ, Min YH, Liu YX, Gao BQ, Liu XQ, Huang Y, Wang H, Yang L, 
            Liu ZJ, Chen LL
  TITLE     CRISPR-array-mediated imaging of non-repetitive and multiplex 
            genomic loci in living cells.
  JOURNAL   Nat Methods. 2024 Sep;21(9):1646-1657. doi: 
            10.1038/s41592-024-02333-3. Epub 2024 Jul 4.
  PUBMED    38965442
REFERENCE   2  (bases 1 to 5162)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-024-02333-3"; journalName: "Nat Methods"; date: 
            "2024-09"; volume: "21"; issue: "9"; pages: "1646-1657"
COMMENT     Sequence Label: pGL3-U6-aptamer
FEATURES             Location/Qualifiers
     source          1..5162
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..320
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     72..92
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     243..262
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    338..352
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     misc_RNA        553..571
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_feature    681..798
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        847..1357
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1276..1294
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1379..1978
                     /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(1379..1398)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1875..1895
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2125..2246
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2136..2155)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2190..2209
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2494..2511)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2665..3253)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2745..2764)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3424..4215,4216..4284))
                     /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     4047..4066
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4285..4389)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4416..4871
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4503..4522)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4713..4734
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5002..5050
                     /note="synthetic polyadenylation signal"
     misc_feature    5064..5155
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5104..5123
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
       61 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      301 tgtggaaagg acgaaacacc gtaatttcta ctaggtggcc ctgaagaagg gcctaagtgt
      361 agattgtaca gtatataata tacataattt ctactaggtg gctcgttgag ctcattagct
      421 ccgagcctaa gtgtagatat tccattccat tccaatccat gtaatttcta ctaggtggag
      481 cagacgatat ggcgtcgctc ctaagtgtag attaaagtct gcaagtggat attaatttct
      541 actaggtggc caacatgagg atcacccatg tctgcagggc ctaagtgtag atattcgagt
      601 ccattcgatg attaatttct actaggtgta gatttttttg aattctcgac ctcgagacaa
      661 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
      721 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
      781 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggccgcgg
      841 ctcgaggggg ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg
      901 ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggactc gcacattctt
      961 cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac
     1021 gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg
     1081 acaaacggaa gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat
     1141 ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga
     1201 gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct
     1261 gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg
     1321 ctccctcgtt gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat
     1381 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     1441 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     1501 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     1561 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     1621 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     1681 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     1741 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     1801 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     1861 aacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     1921 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg
     1981 cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcggtacc
     2041 tttaagacca atgacttaca aggcagctgt agatcttagc cactttctag agtcggggcg
     2101 gccggccgct tcgagcagac atgataagat acattgatga gtttggacaa accacaacta
     2161 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     2221 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     2281 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa
     2341 tcgataagga tccgtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg
     2401 tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc
     2461 gtaggacagg tgccggcagc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     2521 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     2581 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     2641 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     2701 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2761 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2821 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     2881 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     2941 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3001 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3061 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3121 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3181 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3241 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3301 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     3361 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3421 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3481 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3541 ccccagtgct gcaatgatac cgcgggaccc acgctcaccg gctccagatt tatcagcaat
     3601 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3661 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3721 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3781 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3841 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3901 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3961 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4021 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4081 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4141 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4201 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4261 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     4321 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4381 ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag
     4441 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     4501 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     4561 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     4621 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     4681 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     4741 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     4801 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     4861 gcttacaatt tgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     4921 ggcctcttcg ctattacgcc agcccaagct accatgataa gtaagtaata ttaaggtacg
     4981 ggaggtactt ggagcggccg caataaaata tctttatttt cattacatct gtgtgttggt
     5041 tttttgtgtg aatcgatagt actaacatac gctctccatc aaaacaaaac gaaacaaaac
     5101 aaactagcaa aataggctgt ccccagtgca agtgcaggtg ccagaacatt tctctatcga
     5161 ta
//
