LOCUS       CMV-enAsCas12f                4776 bp ds-DNA     circular SYN 09-AUG-2023
DEFINITION  Expression of enAsCas12f in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    CMV-enAsCas12f
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4776)
  AUTHORS   Wu T, Liu C, Zou S, Lyu R, Yang B, Yan H, Zhao M, Tang W
  TITLE     An engineered hypercompact CRISPR-Cas12f system with boosted 
            gene-editing activity.
  JOURNAL   Nat Chem Biol. 2023 Jul 3. doi: 10.1038/s41589-023-01380-9.
  PUBMED    37400536
REFERENCE   2  (bases 1 to 4776)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 9, 2023 from SnapGene Server 1.1.58
            //gt12365.cn
FEATURES             Location/Qualifiers
     source          1..4776
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(59..919)
                     /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     682..701
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(920..1024)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1103..1122)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1294..1673
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1674..1877
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1828..1848
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1919..1938
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1919..1937
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1987..2007
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3304..3324
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3333..3350
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3373..3390)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3379..3603
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3674..3690)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3674..3690)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3687..3709)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3698..3714
                     /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(3722..3752)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3767..3788
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3905..3922)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4076..4664)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4156..4175)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
       61 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
      121 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
      181 gtgctgcaat gataccgcgg ctaccacgct caccggctcc agatttatca gcaataaacc
      241 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
      301 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
      361 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
      421 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
      481 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
      541 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
      601 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
      661 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
      721 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
      781 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
      841 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
      901 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
      961 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     1021 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg
     1081 atcccctagg gtcgactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     1141 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     1201 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     1261 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
     1321 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     1381 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     1441 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     1501 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     1561 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     1621 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
     1681 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
     1741 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
     1801 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
     1861 tctatataag cagagctggt ttagtgaacc gtcagatccg ctagagatcc gcggccgcta
     1921 atacgactca ctatagggag agccgccacc atgaaacgga cagccgacgg aagcgagttc
     1981 gagtcaccaa agaagaagcg gaaagtcatg atcaaggtgt acagatacga gatcgtgaag
     2041 cccctggacc tggattggaa ggaattcggc accatcctga gacagctgca acaagaaacc
     2101 agattcgccc tgaacaaggc cacacagctg gcctgggagt ggatgggctt cagcagcgac
     2161 tacaaggaca accatggcga gtaccctaag agcaaggaca tcctgggcta caccaatgtg
     2221 cacggctacg cctaccacac aatcaaaaca aaggcctaca gactgaacag cggcaatctg
     2281 tcccagacca tcaagcgggc aaccgaccgg tttaaggctt atcagaaaga aatcctgcgg
     2341 ggcgacatgt ccatccccag ctacaagcgg gacattcctc tggacctgat caaggagaac
     2401 atctctgtga acagaatgaa ccacggcgat tacatcgcca gcctgagcct gctgagcaac
     2461 cccgccaagc aggagatgaa cgtgaagcgg aagatcagcg tgatcatcat agttcggggt
     2521 gctggaaaga ccatcatgga cagaatcctc agcggcgaat accaggtgtc cgcctctcag
     2581 atcatccacg acaagagaaa gaagaaatgg tacctgaata tttcttatga tttcgagcct
     2641 cagacaagag tgctggatct caacaagatt atgggcatcg acctgggcgt ggccgtggct
     2701 gtgtacatgg cttttcagca cacccctgcc agatacaagc tggaaggcgg agagatcgag
     2761 aacttccgta ggcaggtcga gtcccgcaga atcagcatgc tgcggcaggg caagtacgcc
     2821 ggcggcgcca gaagaggcca cggcagagat aagcggatca agcctatcga gcagctgaga
     2881 gataagatcg ctaacttcag agacaccacc aaccacagat acagccggta catcgtcgac
     2941 atggccatca aggagggctg cggcacaatc cagatggaag atctgaccgg catcagagat
     3001 attggatcta gattcctgca gaactggacc tactacgacc tgcagcagaa gatcatctac
     3061 aaagccgagg aagccggcat caaagtgatc aaaattagac ctcaatacac aagccagcgg
     3121 tgcagcgagt gcggcaacat cgacagcgga aatagaatcg gccaggccat cttcaagtgc
     3181 agagcgtgtg gatatgaggc caatgccgac tacaacgccg cccgcaacat cgctatccca
     3241 aacatcgaca agatcatcgc cgaaagcatc aaaaaaagaa ccgccgacgg cagcgaattc
     3301 gagcccaaga agaagaggaa agtctaaccg gtcatcatca ccatcaccat tgagtttaaa
     3361 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3421 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3481 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3541 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3601 tggcttctga ggcggaaaga accagctggg gctcgatacc gtcgacctct agctagagct
     3661 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     3721 acaacatacg agccggaagc ataaagtgta aagcctaggg tgcctaatga gtgagctaac
     3781 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3841 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3901 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3961 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4021 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4081 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4141 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4201 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4261 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4321 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4381 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4441 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4501 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4561 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4621 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4681 tttctacggg gtctgacact cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4741 gattatcaaa aaggatcttc acctagatcc ttttaa
//