LOCUS       sequence-422139-        3286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of tandem pre-sgRNA array hcr5 for LbCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3286)
  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 3286)
  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-hcr5
FEATURES             Location/Qualifiers
     source          1..3286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(34..102,103..894)
                     /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(252..271)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1065..1653
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1554..1573
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1807..1824
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1941..1962
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1977..1982,1983..2000,2001..2007)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2015..2031
                     /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     2020..2042
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2039..2055
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2039..2055
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2144..2392
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2144..2164
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2315..2334
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2725..2742)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2725..2741)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2734..2756)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2950..2969)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3069..3091
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3129..3147)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3215..3286,1..33)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
       61 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
      121 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
      181 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
      241 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
      301 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
      361 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
      421 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
      481 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
      541 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
      601 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
      661 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
      721 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
      781 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
      841 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
      901 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
      961 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     1021 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     1081 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     1141 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     1201 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     1261 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     1321 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     1381 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     1441 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     1501 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     1561 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     1621 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     1681 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     1741 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     1801 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     1861 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     1921 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     1981 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     2041 ggaaacagct atgaccatga ttacgaattc gagctcggta cccggggatc ctctagagat
     2101 tccaattcag tcgactggat ccggtaccaa ggtcgggcag gaagagggcc tatttcccat
     2161 gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt
     2221 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     2281 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     2341 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgaatttct
     2401 actaagtgta gatccttaat cctctcccag acataaaatt tctactaagt gtagatgttt
     2461 aaacacaccg ggttaataaa tttctactaa gtgtagatct aggaccagcc tgagagagtt
     2521 gaatttctac taagtgtaga tggaaaattc cactgtaaac tcgtaatttc tactaagtgt
     2581 agatctgact gctcactaga tgttgaatat cgcggctcgt tggtctaggg gtatgattct
     2641 cgcttagggt gcgagaggtc ccgggttcaa atcccggacg agcccttttt tttaatcgtc
     2701 gacctgcagg catgcaagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa
     2761 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     2821 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     2881 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
     2941 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     3001 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3061 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3121 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     3181 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3241 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctga
//
