LOCUS       sequence-478724-        5732 bp    DNA     circular SYN 01-DEC-2025
DEFINITION  Overexpression of dLbCas12aRVRR in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5732)
  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 5732)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 1, 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: pHAGE-IRES-puro-NLS-dLbCas12aRVRR-EGFP-NLS-3xFlag
FEATURES             Location/Qualifiers
     source          1..5732
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(820..839)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(821..839)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(912..929)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(912..928)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(921..943)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1141..1596
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1228..1247)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1438..1459
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1613..1632)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1732..1754
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1792..1810)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1878..1982
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1983..2051,2052..2843)
                     /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(2201..2220)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3014..3602
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3503..3522
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    3890..3911
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3926..3931,3932..3949,3950..3956)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3964..3980
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3969..3991
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3988..4004
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3988..4004
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ccaccgtgag gtggggtacc tccctggcat tcctgggtgg tcttgaggtg tcaccaaagt
       61 acgggcgttg ggcacaccaa ccttacccca ccggttcacc aaacatgccc cggacaagcc
      121 agaagacgac tacacgcact ggcccgtcca ttactggact ggtctgggac agcggaacat
      181 cgtctcagat atgctgacca agcacacaca tgagaggata ctagggtgag ttggggaaga
      241 gcactcccgc agcgcgcatt tgggggaggt tggcaccaag cagagccccg ctagaattca
      301 ctagtaggga tactcctgaa ggcggaaaga ccgggtgcct ttgataacct gtctgggtcc
      361 ccacagttgc cacgtgagca gcttccgcgg gggatcagga tgcttaccta ccaaagcact
      421 gtagttataa ggggagggaa ggggtgggcc tcgcgagagg ccgtagggcg ggaaaacccg
      481 cccccatgtg agtgtagctg tttgttcaac acatgcacca ctcaatgcac atgcgagtaa
      541 gcgtaatggt tgcaggggaa gaaaagggaa aattgggggt gaggacagat gatggcattc
      601 ttgctgtggg tgggccgaag ccgtccacaa gcccgcctgc catatcaacg gattttcatt
      661 ctcttgcagc tttcgctact gcctattggc tttgagaatt ggactctctc tttaccctcg
      721 tcttgacgtt agggtagctc ccgtcgagtc tctgcacctt ccgattagtt gtaagtcatc
      781 tattgttgac cagtggacaa tcgacggtcg agggtctccc cctatagtga gtcgtattaa
      841 tttcgcggga tcgagatctc gatcctctac gccggacgca tcgtggccgg gatcaccggc
      901 gccacgaatt cactggccgt cgttttacaa cgtcgtgact gggaaaacac tggcgttacc
      961 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     1021 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg
     1081 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     1141 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1201 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1261 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1321 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     1381 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1441 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     1501 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     1561 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     1621 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     1681 gacgggcttg tctgctccag gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     1741 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     1801 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     1861 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     1921 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     1981 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     2041 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     2101 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     2161 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     2221 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     2281 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     2341 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     2401 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     2461 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     2521 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     2581 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     2641 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     2701 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     2761 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     2821 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     2881 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     2941 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3001 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3061 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3121 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     3181 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3241 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3301 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3361 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3421 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcatggtc ggaacaggag
     3481 agcgcacgag ggagcttcca gtgggaaacg cctggtatct ttatagtcct gtcgggtttc
     3541 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3601 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3661 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3721 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagttagc gaggaagcgg
     3781 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     3841 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     3901 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     3961 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     4021 ttctagatat gctgttatcc gtcgattgtc cactggtctt actaattact acttcggctt
     4081 ggctcaggat tgccttgtct ataactagat ttaggtttcc ctgagtttga gagcatccac
     4141 ttaaagaatt tcttctttaa ggctcaatta tttaagtccg tagcgtcttc acttgcccac
     4201 acagaagtcc tggaagatga tgtccaggat ctcctcggta ccccctccac ctgtgagccg
     4261 ggtcaggtga ccccgggcca cccgcagcgc ctctgccgcc agggccaggt cttttgactg
     4321 cttgtagtgg ccgagggcat ccaggcaacc ctggaggtgg tgctggtgcc ttgctcgggt
     4381 cagcagcggg ggatctgtgg acgggtcccc acacactgca gctagctcct tcctcagcgc
     4441 ctccaggagg ccgtccagcc cctctcccgt cagacaggac agcagcaggt gcgggggcag
     4501 gtcaggaccg ggacctgggc cctccgggga cagcaggtcc gacttgttca gcaccaggag
     4561 gaggcgctgg ctgctgtcac tggggctctg ggctcccaca gaggctacga cggtggccag
     4621 gaagttgcaa ctggagggag aggccaggtc agaagcatcc agcatggcca gaatgaggtc
     4681 agcctgctct agcctctccc gggcgcgccg cacgccctcc tgctccacgg gccccacgcc
     4741 ctcccgcaac ccagccgtgt cgctcagcag cacaggaaat ccggccaggt cgactggggt
     4801 ctccagcacg tcacgggtgg tccctggctc cggggacacg atggacacag gcttccgact
     4861 gagcaggttc actaggctgc tcttgcccgc attggggggt ccagtgacca ctacgtgcgc
     4921 ccctgagcgg agcctctgcc cgcgcctggc atctcgtaga tgtgcaccca gggccacctg
     4981 cagtgcccgt acttcgatgt cggcttgctc caggaccccc tcctccaggt tttcatcctc
     5041 gccgaaatcg atataggcct ccacgtgggc cagagctttg gtgagggtct cggcccagcc
     5101 acggcagagg tggcccagct ctccgtccag ctgcctgagg gcctgccgcc gctgcgcctc
     5161 tgtttccgcg tggataaggt ccgccagccc ctccacttcg gtcaggttca gcttcccatt
     5221 tgcgaacgcc cgtctggtga actcgcctgc ctccgccggt cgaagccctg gcacgctgcc
     5281 caaggcctgc aggacgccgc tcaccactgc cgggcctcca tgcacgtgga actccacgca
     5341 gtcctcaccg gtgaaactct ggggacctgg gaaccagagc accagtgcgc ggtccagagg
     5401 ctccccggag cggggatcgc tgagcaggcg caggctggcg tggcgagcaa ggggcaggtc
     5461 tcggggtgcg gtgagaattc ggagggcgtg gccgctggcg gggccgctgg tccggatcac
     5521 tgcgatgccg cagcggcctt ggccagagct tagcgcgaag atggtggcgc cggagccggg
     5581 ggctggtgcg ccgctgctcc ggcgcgtgca caatctgcga ggcccacgtg ccgcttgggc
     5641 cgccagggtc caaagccccc gccacatcgt gaacagaaga aagaagtgga agcaagaaga
     5701 accaccgtag acaattagcc cagggtcaga tc
//
