LOCUS       sequence-274024-        8166 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dLbCas12a-RVRR-BE in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8166)
  AUTHORS   Toth E, Varga E, Kulcsar PI, Kocsis-Jutka V, Krausz SL, Nyeste A, 
            Welker Z, Huszar K, Ligeti Z, Talas A, Welker E
  TITLE     Improved LbCas12a variants with altered PAM specificities further 
            broaden the genome targeting range of Cas12a nucleases.
  JOURNAL   Nucleic Acids Res. 2020 Feb 28. pii: 5763098. doi: 
            10.1093/nar/gkaa110.
  PUBMED    32107556
REFERENCE   2  (bases 1 to 8166)
  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: "Nucleic 
            Acids Res. 2020 Feb 28. pii: 5763098. doi: 10.1093/nar/gkaa110."
COMMENT     Sequence Label: pTE3987
FEATURES             Location/Qualifiers
     source          1..8166
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             412..432
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             433..1119
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             4858..4878
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4891..5139
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5152..5172
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5181..5198
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5221..5238)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5227..5451
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5522..5538)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5522..5538)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5535..5557)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5546..5562
                     /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(5570..5576,5577..5594,5595..5600))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5615..5636
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5753..5770)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5924..6512)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6004..6023)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6683..7474,7475..7543))
                     /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     7306..7325
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7544..7648)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7727..7746)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7918..8166,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gcccaagaag
      421 aagaggaaag tcatgagctc agagactggc ccagtggctg tggaccccac attgagacgg
      481 cggatcgagc cccatgagtt tgaggtattc ttcgatccga gagagctccg caaggagacc
      541 tgcctgcttt acgaaattaa ttgggggggc cggcactcca tttggcgaca tacatcacag
      601 aacactaaca agcacgtcga agtcaacttc atcgagaagt tcacgacaga aagatatttc
      661 tgtccgaaca caaggtgcag cattacctgg tttctcagct ggagcccatg cggcgaatgt
      721 agtagggcca tcactgaatt cctgtcaagg tatccccacg tcactctgtt tatttacatc
      781 gcaaggctgt accaccacgc tgacccccgc aatcgacaag gcctgcggga tttgatctct
      841 tcaggtgtga ctatccaaat tatgactgag caggagtcag gatactgctg gagaaacttt
      901 gtgaattata gcccgagtaa tgaagcccac tggcctaggt atccccatct gtgggtacga
      961 ctgtacgttc ttgaactgta ctgcatcata ctgggcctgc ctccttgtct caacattctg
     1021 agaaggaagc agccacagct gacattcttt accatcgctc ttcagtcttg tcattaccag
     1081 cgactgcccc cacacattct ctgggccacc gggttgaaaa gcggcagcga gactcccggg
     1141 acctcagagt ccgccacacc cgaaagtatg tcaaagctgg agaaattcac caactgttat
     1201 agcctgtcta agaccctgcg cttcaaggca atcccagtgg gcaagacaca agagaacatt
     1261 gacaacaaac ggctcctggt ggaggatgag aagagggctg aagattacaa gggcgttaag
     1321 aagctgctgg ataggtacta tctgtcattc atcaacgatg tcctccacag tatcaagctg
     1381 aagaatctga acaattacat ttctctgttc cggaagaaga cacggaccga gaaggagaac
     1441 aaagagctgg agaatctgga gatcaacctg aggaaagaaa tagctaaggc tttcaaaggg
     1501 aacgagggtt acaagtccct gttcaagaaa gacattatcg agactattct gcctgagttc
     1561 ctggacgata aagatgagat cgccctcgtc aattccttca atgggtttac cacagccttt
     1621 accggcttct tcgacaatag agagaatatg ttctctgaag aggccaaatc cactagcatc
     1681 gcctttcgct gcataaacga gaacctgact aggtacatca gcaatatgga catctttgag
     1741 aaagtcgatg ccatattcga caaacatgag gtgcaggaga ttaaggagaa gatcctgaac
     1801 tcagattacg atgtcgaaga tttcttcgag ggagagttct tcaacttcgt gctcacacaa
     1861 gagggcattg atgtgtacaa tgcaatcatt ggagggttcg tgacagagag tggcgagaag
     1921 ataaagggcc tgaacgagta tatcaacctc tacaaccaga aaaccaagca gaaactgcct
     1981 aagttcaagc cactgtacaa acaagtgctc tcagataggg aaagcctgag cttctacggt
     2041 gaagggtata catcagatga agaagtgctc gaagtgttcc gcaacaccct caataagaac
     2101 agtgaaatct tctcttcaat caagaagctg gagaaactgt tcaagaattt cgatgagtac
     2161 tcctctgccg gaatctttgt gaagaatggc cctgcaatat ccactattag caaagacatc
     2221 tttggcgagt ggaacgttat cagggataag tggaatgccg agtacgatga tattcatctc
     2281 aagaagaaag ccgtggttac agagaaatac gaggatgata gacgcaagag ctttaagaag
     2341 attggtagct tctctctcga acagctgcag gagtacgccg acgctgacct gtcagtcgtg
     2401 gagaaactca aggagatcat aatccagaag gtggatgaaa tctacaaagt gtatggaagc
     2461 tctgagaaac tcttcgatgc agactttgtt ctggagaaga gtctgaagaa gaacgacgca
     2521 gtggttgcta tcatgaagga cctgctggat tctgttaagt ctttcgagaa ttacattaag
     2581 gcattctttg gtgaagggaa ggagacaaat agggacgaga gcttctatgg cgactttgtt
     2641 ctggcctacg acatcctcct caaggttgac cacatctatg acgctatacg gaattacgtt
     2701 acccagaagc cctatagcaa agacaagttc aagctgtatt tccagaatcc acagtttatg
     2761 agggggtggg ataaagacgt ggaaacagat aggagggcca ctatcctgcg gtacggcagc
     2821 aaatactatc tggctatcat ggataagaag tacgccaaat gcctccagaa gatcgacaag
     2881 gacgacgtga acggtaacta cgagaagatc aattacaagc tcctgccagg acctaacaag
     2941 atgctgccca gggtgttctt ctccaagaaa tggatggcct actataaccc aagcgaggac
     3001 attcagaaga tatacaagaa tgggacattc aagaagggcg atatgttcaa cctcaacgac
     3061 tgccacaagc tgattgattt cttcaaggat agcatttctc gctatcccaa gtggtctaat
     3121 gcatacgatt tcaacttcag cgagactgag aagtacaaag acatcgctgg cttctaccgg
     3181 gaggtggaag agcaaggcta taaggtgtca ttcgaatccg cttctaagaa ggaagtggat
     3241 aagctcgtgg aagagggtaa gctgtacatg ttccagatat acaacaaaga cttcagcgat
     3301 aagagccacg gcactccaaa cctccatact atgtatttca agctgctgtt tgacgagaac
     3361 aaccacggac agattaggct gtcaggaggc gcagaactct tcatgcgcag agcttcactg
     3421 aagaaggagg aactcgttgt ccacccagcc aatagcccta tagccaataa gaatccagac
     3481 aatcctaaga aaaccactac tctgtcttac gatgtgtata aggataagag attctctgaa
     3541 gatcagtacg aactgcacat acccattgcc attaacaagt gccctaagaa catcttcaag
     3601 attaacacag aggttagagt gctcctgaaa cacgacgata acccttatgt tataggcatt
     3661 gctcgcggag agagaaacct gctgtacatc gtcgtggtgg acggcaaagg caacatcgtg
     3721 gaacagtaca gtctcaatga aatcattaac aatttcaacg gaatccgcat taagaccgac
     3781 taccattctc tcctcgacaa gaaggagaaa gaaaggttcg aagcaagaca gaattggaca
     3841 agtatagaga atatcaaaga actgaaggct gggtacatct ctcaggttgt gcacaagata
     3901 tgtgagctgg tggagaagta cgacgctgtt atcgccctcg cggacctgaa tagcggcttc
     3961 aagaactcca gggtgaaggt ggagaagcag gtgtatcaga agttcgagaa gatgctgatc
     4021 gacaagctca actatatggt ggacaagaaa tccaatcctt gcgctactgg tggagccctg
     4081 aagggctatc aaatcaccaa taagttcgaa tctttcaagt ctatgagcac ccagaatggc
     4141 ttcatcttct acatacccgc atggctgaca tccaagattg atccctctac cggatttgtt
     4201 aatctgctca agactaagta cacctctatt gctgactcaa agaagttcat atcatcattt
     4261 gaccgcatca tgtacgtgcc agaagaggac ctgttcgagt ttgccctgga ttacaagaat
     4321 ttctctcgga ctgacgccga ctacatcaag aagtggaagc tctactctta tggtaatcgg
     4381 attcgcatat tccgcaatcc caagaagaat aacgtgttcg attgggagga agtttgcctc
     4441 accagcgctt acaaggagct gttcaataag tatgggatta actaccagca gggcgacata
     4501 agagccctgc tgtgcgaaca atctgataag gcattctatt cctctttcat ggcactgatg
     4561 tcactgatgc tgcaaatgcg caattccatc accggaagaa cagacgtggc ctttctgatc
     4621 tctcctgtca agaactcaga tggcatcttc tacgattccc gcaactatga agcacaggag
     4681 aatgctatcc tgcctaagaa tgccgatgca aatggagcct ataacatcgc cagaaaggtc
     4741 ctctgggcca taggacaatt caagaaagct gaagatgaga agctggacaa ggtgaagatc
     4801 gccatttcaa acaaagagtg gctcgaatat gctcagacct cagtgaagca tggatcaccc
     4861 aagaagaaac ggaaagtgtc tggtggttct actaatctgt cagatattat tgaaaaggag
     4921 accggtaagc aactggttat ccaggaatcc atcctcatgc tcccagagga ggtggaagaa
     4981 gtcattggga acaagccgga aagcgatata ctcgtgcaca ccgcctacga cgagagcacc
     5041 gacgagaatg tcatgcttct gactagcgac gcccctgaat acaagccttg ggctctggtc
     5101 atacaggata gcaacggtga gaacaagatt aagatgctct ctggtggttc tcccaagaag
     5161 aagaggaaag tctaaccggt catcatcacc atcaccattg agtttaaacc cgctgatcag
     5221 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     5281 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     5341 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     5401 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     5461 cggaaagaac cagctggggc tcgataccgt cgacctctag ctagagcttg gcgtaatcat
     5521 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     5581 ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc acattaattg
     5641 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5701 tcggccaacg cgcgggaaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5761 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5821 taccacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5881 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5941 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6001 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6061 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6121 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6181 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6241 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6301 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6361 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6421 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6481 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6541 ctgacgctca gtggaacgaa aacgtacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6601 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6661 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6721 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6781 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6841 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6901 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6961 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7021 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7081 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7141 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7201 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7261 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7321 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7381 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7441 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7501 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7561 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7621 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     7681 acggatcggg agatcgatct cccgatcccc tagggtcgac tctcagtaca atctgctctg
     7741 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     7801 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     7861 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
     7921 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     7981 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     8041 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     8101 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     8161 tgtatc
//
