LOCUS       sequence-482726-        5732 bp    DNA     circular SYN 08-AUG-2025
DEFINITION  Overexpression of dLbCas12a 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 Aug 8, 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-dLbCas12a-EGFP-NLS-3xFlag
FEATURES             Location/Qualifiers
     source          1..5732
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2201..2217)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2201..2217)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2214..2236)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2225..2241
                     /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)."
     promoter        complement(join(2249..2255,2256..2273,2274..2279))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2294..2315
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2603..3191)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2683..2702)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3362..4153,4154..4222))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTSLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3985..4004
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4223..4327)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4395..4413
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4451..4473)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4573..4592
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4609..5064)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4746..4767)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4958..4977
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5262..5284
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5276..5293
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5277..5293
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5366..5385
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5366..5384
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 cccgccctac ggcctctcgc gaggcccacc ccttccctcc ccttataact acagtgcttt
       61 ggtaggtaag catcctgatc ccccgcggaa gctgctcacg tggcaactgt ggggacccag
      121 acaggttatc aaaggcaccc ggtctttccg ccttcaggag tatccctact agtgaattct
      181 agcggggctc tgcttggtgc caacctcccc caaatgcgcg ctgcgggagt gctcttcccc
      241 aactcaccct agtatcctct catgtgtgtg cttggtcagc atatctgaga cgatgttccg
      301 ctgtcccaga ccagtccagt aatggacggg ccagtgcgtg tagtcgtctt ctggcttgtc
      361 cggggcatgt ttggtgaacc ggtggggtaa ggttggtgtg cccaacgccc gtactttggt
      421 gacacctcaa gaccacccag gaatgccagg gaggtacccc acctcacggt gggatctgac
      481 cctgggctaa ttgtctacgg tggttcttct tgcttccact tctttcttct gttcacgatg
      541 tggcgggggc tttggaccct ggcggcccaa gcggcacgtg ggcctcgcag attgtgcacg
      601 cgccggagca gcggcgcacc agcccccggc tccggcgcca ccatcttcgc gctaagctct
      661 ggccaaggcc gctgcggcat cgcagtgatc cggaccagcg gccccgccag cggccacgcc
      721 ctccgaattc tcaccgcacc ccgagacctg ccccttgctc gccacgccag cctgcgcctg
      781 ctcagcgatc cccgctccgg ggagcctctg gaccgcgcac tggtgctctg gttcccaggt
      841 ccccagagtt tcaccggtga ggactgcgtg gagttccacg tgcatggagg cccggcagtg
      901 gtgagcggcg tcctgcaggc cttgggcagc gtgccagggc ttcgaccggc ggaggcaggc
      961 gagttcacca gacgggcgtt cgcaaatggg aagctgaacc tgaccgaagt ggaggggctg
     1021 gcggacctta tccacgcgga aacagaggcg cagcggcggc aggccctcag gcagctggac
     1081 ggagagctgg gccacctctg ccgtggctgg gccgagaccc tcaccaaagc tctggcccac
     1141 gtggaggcct atatcgattt cggcgaggat gaaaacctgg aggagggggt cctggagcaa
     1201 gccgacatcg aagtacgggc actgcaggtg gccctgggtg cacatctacg agatgccagg
     1261 cgcgggcaga ggctccgctc aggggcgcac gtagtggtca ctggaccccc caatgcgggc
     1321 aagagcagcc tagtgaacct gctcagtcgg aagcctgtgt ccatcgtgtc cccggagcca
     1381 gggaccaccc gtgacgtgct ggagacccca gtcgacctgg ccggatttcc tgtgctgctg
     1441 agcgacacgg ctgggttgcg ggagggcgtg gggcccgtgg agcaggaggg cgtgcggcgc
     1501 gcccgggaga ggctagagca ggctgacctc attctggcca tgctggatgc ttctgacctg
     1561 gcctctccct ccagttgcaa cttcctggcc accgtcgtag cctctgtggg agcccagagc
     1621 cccagtgaca gcagccagcg cctcctcctg gtgctgaaca agtcggacct gctgtccccg
     1681 gagggcccag gtcccggtcc tgacctgccc ccgcacctgc tgctgtcctg tctgacggga
     1741 gaggggctgg acggcctcct ggaggcgctg aggaaggagc tagctgcagt gtgtggggac
     1801 ccgtccacag atcccccgct gctgacccga gcaaggcacc agcaccacct ccagggttgc
     1861 ctggatgccc tcggccacta caagcagtca aaagacctgg ccctggcggc agaggcgctg
     1921 cgggtggccc ggggtcacct gacccggctc acaggtggag ggggtaccga ggagatcctg
     1981 gacatcatct tccaggactt ctgtgtgggc aagtgaagac gctacggact taaataattg
     2041 agccttaaag aagaaattct ttaagtggat gctctcaaac tcagggaaac ctaaatctag
     2101 ttatagacaa ggcaatcctg agccaagccg aagtagtaat tagtaagacc agtggacaat
     2161 cgacggataa cagcatatct agaagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     2221 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     2281 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     2341 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     2401 gcggtttgcg tattgggcgc tcttccgctt cctcgctaac tgactcgctg cgctcggtcg
     2461 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     2521 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     2581 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     2641 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     2701 ccactggaag ctccctcgtg cgctctccta ttccgaccat gccgcttacc ggatacctgt
     2761 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     2821 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     2881 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     2941 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3001 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     3061 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3121 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3181 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3241 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     3301 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     3361 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     3421 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     3481 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     3541 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     3601 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     3661 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     3721 tcagctccgg ttcccaacga tcaaggctag ttacatgatc ccccatgttg tgcaaaaaag
     3781 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     3841 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     3901 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     3961 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4021 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4081 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4141 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcta
     4201 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     4261 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     4321 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     4381 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     4441 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     4501 atgcctggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     4561 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccataaa attgtaaacg
     4621 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     4681 agaccgaaat cggcaaaatc ccttataaat caaaagaata gcccgagata gagttgagtg
     4741 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     4801 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcacccaaa tcaagttttt
     4861 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     4921 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     4981 gcgctaaggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     5041 ttaatgcgcc gctacagggc gcgtactatg gttgctttga cgtatgcggt gtgaaatacc
     5101 gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa
     5161 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     5221 atgtgctgca aggcgattaa gttgggtaac gccagtgttt tcccagtcac gacgttgtaa
     5281 aacgacggcc agtgaattcg tggcgccggt gatcccggcc acgatgcgtc cggcgtagag
     5341 gatcgagatc tcgatcccgc gaaattaata cgactcacta tagggggaga ccctcgaccg
     5401 tcgattgtcc actggtcaac aatagatgac ttacaactaa tcggaaggtg cagagactcg
     5461 acgggagcta ccctaacgtc aagacgaggg taaagagaga gtccaattct caaagccaat
     5521 aggcagtagc gaaagctgca agagaatgaa aatccgttga tatggcaggc gggcttgtgg
     5581 acggcttcgg cccacccaca gcaagaatgc catcatctgt cctcaccccc aattttccct
     5641 tttcttcccc tgcaaccatt acgcttactc gcatgtgcat tgagtggtgc atgtgttgaa
     5701 caaacagcta cactcacatg ggggcgggtt tt
//
