LOCUS       sequence-478947-        5753 bp    DNA     circular SYN 17-JUL-2025
DEFINITION  Overexpression of dLbCas12a in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5753)
  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 5753)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 17, 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..5753
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2044..2063)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2045..2063)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2136..2153)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2136..2152)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2145..2167)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2365..2820
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2452..2471)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2662..2683
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2837..2856)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2956..2978
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3016..3034)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3102..3206
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3207..3275,3276..4067)
                     /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(3425..3444)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4238..4826
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4727..4746
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4980..4997
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5114..5135
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5150..5155,5156..5173,5174..5180)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5188..5204
                     /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     5193..5215
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5212..5228
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5212..5228
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 acagcaggtc cgacttgttc agcaccagga ggaggcgctg gctgctgtca ctggggctct
       61 gggctcccac agaggctacg acggtggcca ggaagttgca actggaggga gaggccaggt
      121 cagaagcatc cagcatggcc agaatgaggt cagcctgctc tagcctctcc cgggcgcgcc
      181 gcacgccctc ctgctccacg ggccccacgc cctcccgcaa cccagccgtg tcgctcagca
      241 gcacaggaaa tccggccagg tcgactgggg tctccagcac gtcacgggtg gtccctggct
      301 ccggggacac gatggacaca ggcttccgac tgagcaggtt cactaggctg ctcttgcccg
      361 cattgggggg tccagtgacc actacgtgcg cccctgagcg gagcctctgc ccgcgcctgg
      421 catctcgtag atgtgcaccc agggccacct gcagtgcccg tacttcgatg tcggcttgct
      481 ccaggacccc ctcctccagg ttgtcatcct cgccgaaatc gatataggcc tccacgtggg
      541 ccagagcttt ggtgagggtc tcggcccagc cacggcagag gtggcccagc tctccgtcca
      601 gctgcctgag ggcctgccgc cgctgcgcct ctgtttccgc gtggataagg tccgccagcc
      661 cctccacttc ggtcaggttc agcttcccat tggcgaacgc ccgtctggtg aactcgcctg
      721 cctccgccgg tcgaagccct ggcacgctgc ccaaggcctg caggacgccg ctcaccactg
      781 ccgggcctcc atgcacgtgg aactccacgc agtcctcacc ggtgaaactc tggggacctg
      841 ggaaccagag caccagtgcg cggtccagag gctccccgga gcggggatcg ctgagcaggc
      901 gcaggctggc gtggcgagca aggggcaggt ctcggggtgc ggtgagaatt cggagggcgt
      961 ggccgctggc ggggccgctg gtccggatca ctgcgatgcc gcagcggcct tggccagagc
     1021 ttagcgcgaa gatggtggcg ccggagccgg gggctggtgc gccgctgctc cggcgcgtgc
     1081 acaatctgcg aggcccacgt gccgcttggg ccgccagggt ccaaagcccc cgccacatca
     1141 tgccagaaaa aagagaagtg gaagcaagaa gaaccaccgt agacaattag cccagggtca
     1201 gatcccaccg aagtggggta cctccctggc attcctgggt ggtcttgagg tatcaccaaa
     1261 ttacgggcgt tgggcacacc aaccttacct cagcgtttcg ccaaacatgc gccggaaaac
     1321 cggaaagcga ctacgcacac tggcccgtcc attgctggac tggtctggga cagcggaacg
     1381 tcgcctcaga tatgctgacc aagcacacac atgagaggtt actaagatga gttggggaag
     1441 agcactcccg cagcgcgcat ttgggggagg ttggcaccaa gcagagccct accggaattc
     1501 accggcagag atactcctga aggcggaaag accgggtgcc tttgataacc tgcctggatc
     1561 cccacagttg ccacgttagc agcttccgcg gggggtcagg gtgctcacct accaaagcac
     1621 tgtagttatg ggaagtgagg gcggatagaa agagagagag ggaggggggg gttccgcgag
     1681 gaacccgagg gcgggacaaa cccgccccga agggtgtggg tgtagctgtt tggacaacgc
     1741 acgtgccact caacacgtgc gcgagtaagc gtggtggttg cgggggaagg gaaggaatac
     1801 gggtaatgag gacagatgac ggcattcttg tcgcgggtgg gccgaagccg tccgcagacc
     1861 ccccatgttc aacggatttt cattctcttg cagctttcgc tactgcctat tggctttgag
     1921 aattggactc tctctttacc ctcgtcttga cgttagggta gctcccgtcg agtctctgca
     1981 ccttccgatt agttgtaagt catctattgt tgaccagtgg acaatcgacg gtcgagggtc
     2041 tccccctata gtgagtcgta ttaatttcgc gggatcgaga tctcgatcct ctacgccgga
     2101 cgcatcgtgg ccgggatcac cggcgccacg aattcactgg ccgtcgtttt acaacgtcgt
     2161 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2221 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2281 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2341 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2401 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     2461 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2521 ggctaccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2581 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2641 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     2701 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     2761 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     2821 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     2881 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     2941 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3001 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     3061 tggtttctta gacgttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3121 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3181 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3241 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3301 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3361 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3421 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     3481 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3541 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3601 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3661 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3721 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3781 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3841 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3901 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     3961 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     4021 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4081 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4141 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4201 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4261 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4321 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4381 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4441 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4501 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4561 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4621 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4681 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4741 atctttatag tcctgtcggg tttcgccacc tctggcttga gcgtcgattt ttgtgatgct
     4801 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4861 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4921 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4981 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     5041 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     5101 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     5161 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     5221 gctatgacca tgattacgcc aagcttctag atatgctgtt atccgtcgat tgtccactgg
     5281 tcttactaat tactacttcg gcttggctca ggattgcctt gtctataact agatttaggt
     5341 ttccctgagt ttgagagcat ccacttaaag aatttcttct ttaaggctca attatttaag
     5401 tccgtagcgt cttcacttgc ccacacagaa gtcctggaag atgatgtcca ggatctcctc
     5461 ggtaccccct ccacctgtga gccgggtcag gtgaccccgg gccacccgca gcgcctctgc
     5521 cgccagggcc aggtcttttg actgcttgta gtggccgagg gcatccaggc aaccctggag
     5581 gtggtgctgg tgccttgctc gggtcagcag cgggggatct gtggacgggt ccccacacac
     5641 tgcagctagc tccttcctca gcgcctccag gaggccgtcc agcccctctc ccgtcagaca
     5701 gtacagcagc aggtgcgggg gcaggtcagg accgggacct gggccctccg ggg
//
