LOCUS       sequence-226262-        3161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty vector for cloning AsCpf1-SpCas9 fgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3161)
  AUTHORS   Kweon J, Jang AH, Kim DE, Yang JW, Yoon M, Rim Shin H, Kim JS, Kim Y
  TITLE     Publisher Correction: Fusion guide RNAs for orthogonal gene 
            manipulation with Cas9 and Cpf1.
  JOURNAL   Nat Commun. 2018 Jan 16;9(1):303. doi: 10.1038/s41467-017-02686-8.
  PUBMED    29335559
REFERENCE   2  (bases 1 to 3161)
  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: "Nat 
            Commun."; date: "2018-01-16"; volume: "9(1):303. doi"; pages: " 
            10.1038/s41467-017-02686-8"
COMMENT     Sequence Label: pFGRNA-AsCpf1-SpCas9
FEATURES             Location/Qualifiers
     source          1..3161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        84..332
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     84..104
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     255..274
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     427..445
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(483..505)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(600..917)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNRLA
                     AHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDA
                     A"
     primer_bind     605..624
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     818..840
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     832..849
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     833..849
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    853..909
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(918..934)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(918..934)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(931..953)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    942..958
                     /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(966..972,973..990,991..996))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     misc_RNA        1026..1101
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1235..1252)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1406..1994)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1486..1505)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2165..2956,2957..3025))
                     /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     2788..2807
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3026..3130)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgaagact caattgtcga ttagtgaacg gatctcgacg gtatcgatca cgagactagc
       61 ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata
      121 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
      181 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
      241 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
      301 ctttatatat cttgtggaaa ggacgaaaca ccgtaatttc tactcttgta gatggagacc
      361 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
      421 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
      481 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
      541 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
      601 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
      661 agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt
      721 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
      781 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      841 acggccagtg ccaagcttgc atgcctgcag gtcgactcta gaggatcccc gggtaccgag
      901 ctcgaattcg taatcatgtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
      961 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atagatccgg
     1021 tctccgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     1081 aaagtggcac cgagtcggtg cttttttcta gattcgcgat gtacgggcca gatatacgcg
     1141 ttgacattga ttattgacta gttgtcttcc tgcattaatg aatcggccaa cgcgcgggga
     1201 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     1261 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     1321 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     1381 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1441 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1501 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1561 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     1621 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1681 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1741 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1801 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     1861 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1921 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1981 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2041 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2101 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2161 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2221 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2281 gccccagtgc tgcaatgata ccgcgagatc cacgctcacc ggctccagat ttatcagcaa
     2341 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     2401 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     2461 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2521 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2581 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     2641 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     2701 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     2761 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     2821 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     2881 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     2941 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3001 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3061 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3121 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
