LOCUS       sequence-229036-        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        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     344..362
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(400..422)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(517..834)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNRLA
                     AHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDA
                     A"
     primer_bind     522..541
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     735..757
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     749..766
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     750..766
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    770..826
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(835..851)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(835..851)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(848..870)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    859..875
                     /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(883..889,890..907,908..913))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     misc_RNA        943..1018
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1152..1169)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1323..1911)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1403..1422)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2082..2873,2874..2942))
                     /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     2705..2724
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2943..3047)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg taatttctac tcttgtagat ggagaccaca tttccccgaa aagtgccacc
      301 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
      361 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
      421 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
      481 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
      541 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
      601 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
      661 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
      721 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgcca agcttgcatg
      781 cctgcaggtc gactctagag gatccccggg taccgagctc gaattcgtaa tcatgtcata
      841 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
      901 cataaagtgt aaagcctggg gtgcctaata gatccggtct ccgttttaga gctagaaata
      961 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     1021 ttttctagat tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
     1081 gtcttcctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1141 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1201 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1261 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1321 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1381 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1441 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1501 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1561 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1621 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     1681 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     1741 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     1801 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     1861 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     1921 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     1981 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2041 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2101 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2161 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2221 cgagatccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2281 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2341 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2401 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2461 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     2521 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     2581 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     2641 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     2701 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     2761 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     2821 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     2881 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     2941 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3001 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3061 aaagtgccac ctgacgtcga cgaagactca attgtcgatt agtgaacgga tctcgacggt
     3121 atcgatcacg agactagcct cgagcggccg cccccttcac c
//
