LOCUS       sequence-422140-        3200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of tandem pre-sgRNA array hcr3 for LbCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3200)
  AUTHORS   Chen F, Lian M, Ma B, Gou S, Luo X, Yang K, Shi H, Xie J, Ge W, 
            Ouyang Z, Lai C, Li N, Zhang Q, Jin Q, Liang Y, Chen T, Wang J, Zhao
            X, Li L, Yu M, Ye Y, Wang K, Wu H, Lai L
  TITLE     Multiplexed base editing through Cas12a variant-mediated cytosine 
            and adenine base editors.
  JOURNAL   Commun Biol. 2022 Nov 2;5(1):1163. doi: 10.1038/s42003-022-04152-8.
  PUBMED    36323848
REFERENCE   2  (bases 1 to 3200)
  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: "Commun 
            Biol."; date: "2022-11-2"; volume: "5(1):1163. doi"; pages: " 
            10.1038/s42003-022-04152-8"
COMMENT     Sequence Label: U6-sgRNA-hcr3
FEATURES             Location/Qualifiers
     source          1..3200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..37)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      831..1419
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1320..1339
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1573..1590
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1707..1728
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1743..1748,1749..1766,1767..1773)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1781..1797
                     /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     1786..1808
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1805..1821
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1805..1821
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1910..2158
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1910..1930
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2081..2100
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2405..2422)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2405..2421)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2414..2436)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2630..2649)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2749..2771
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2809..2827)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2895..2999
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3000..3068,3069..3200,1..660)
                     /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"
ORIGIN
        1 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
       61 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
      121 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
      181 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
      241 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
      301 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
      361 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
      421 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
      481 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
      541 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
      601 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
      661 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
      721 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
      781 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
      841 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
      901 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
      961 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     1021 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     1081 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     1141 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1201 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1261 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1321 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     1381 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     1441 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     1501 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     1561 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     1621 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     1681 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     1741 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     1801 cacacaggaa acagctatga ccatgattac gaattcgagc tcggtacccg gggatcctct
     1861 agagattcca attcagtcga ctggatccgg taccaaggtc gggcaggaag agggcctatt
     1921 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
     1981 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     2041 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     2101 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga
     2161 atttctacta agtgtagatc tgactgctca ctagatgttg aaaatttcta ctaagtgtag
     2221 atgtttaaac acaccgggtt aataaatttc tactaagtgt agatctagga ccagcctgag
     2281 agagttgtat cgcggctcgt tggtctaggg gtatgattct cgcttagggt gcgagaggtc
     2341 ccgggttcaa atcccggacg agcccttttt tttaatcgtc gacctgcagg catgcaagct
     2401 tggcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     2461 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     2521 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc
     2581 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
     2641 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     2701 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     2761 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     2821 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     2881 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2941 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     3001 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     3061 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     3121 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     3181 aagaacgttt tccaatgatg
//
