LOCUS       sequence-182673-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human FZR1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7302)
  AUTHORS   McKinley KL, Cheeseman IM
  TITLE     Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the
            Diversity of p53-Dependent Responses to Cell-Cycle Defects.
  JOURNAL   Dev Cell. 2017 Feb 27;40(4):405-420.e2. doi: 
            10.1016/j.devcel.2017.01.012. Epub 2017 Feb 16.
  PUBMED    28216383
REFERENCE   2  (bases 1 to 7302)
  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"; doi: 
            "10.1016/j.devcel.2017.01.012"; journalName: "Dev Cell"; date: 
            "2017-02-27- 27"; volume: "40"; issue: "4"; pages: "405-420.e2"
COMMENT     Sequence Label: FZR1 D10.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(126..714)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(206..225)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(885..1676,1677..1745))
                     /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     1508..1527
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1746..1850)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1876..2331)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2013..2034)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2225..2244
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2458..2480
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2472..2489
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2473..2489
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2499..2518
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2499..2517
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      2538..2673
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2713..2834
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2724..2743)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2778..2797
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             2912..3145
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    3217..3805
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3323..3334
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     3732..3752
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3821..4420)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3904..3924)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4401..4420
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4446..4657)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    4726..4843
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(4999..5247)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5057..5076)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5227..5247)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(5425..5469)
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    5654..5887
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    6380..6505
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             6552..6732
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(6733..6959)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(6987..7007)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6987..7005)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(7026..7042)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7026..7042)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7039..7061)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7050..7066
                     /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(7074..7080,7081..7098,7099..7104))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7119..7140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7257..7274)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
       61 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
      121 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      181 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      241 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      301 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      361 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
      421 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
      481 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
      541 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
      601 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
      661 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
      721 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
      781 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
      841 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
      901 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
      961 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     1021 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     1081 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     1141 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     1201 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     1261 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     1321 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     1381 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     1441 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     1501 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     1561 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     1621 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     1681 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     1741 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     1801 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     1861 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     1921 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     1981 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     2041 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     2101 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     2161 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     2221 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     2281 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     2341 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     2401 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     2461 agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg
     2521 aattgggtac gtccctaggc ctccaaaaaa gcctcctcac tacttctgga atagctcaga
     2581 ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga
     2641 atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggatagctag
     2701 agccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     2761 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     2821 gcaataaaca agttcctctc actctctgat attcatttct ttgcaagtta taaatactga
     2881 ataataagat gacatgaact actatacgta ctgctagaga ttttccacac tgactaaaag
     2941 ggtctgaggg atctctagtt accagagtca cacaacagac gggcacacac tacttgaagc
     3001 actcaaggca agctttattg aggcttaagc agtgggttcc ctagttagcc agagagctcc
     3061 caggctcaga tctggtctaa ccagagagac ccagtacaag caaaaagcag atcttgtctt
     3121 cgttgggagt gaattagccc ttccagtccc cccttttctt ttaaaaagtg gctaagatct
     3181 acagctgcct tgtaagtcat tggtcttaaa gtcgacgcgg ggaggcggcc caaagggaga
     3241 tccgactcgt ctgagggcga aggcgaagac gcggaagagg ccgcagagcc ggcagcaggc
     3301 cgcgggaagg aaggtccgct ggattgaggg ccgaagggac gtagcagaag gacgtcccgc
     3361 gcagaatcca ggtggcaaca caggcgagca gccaaggaaa ggacgatgat ttccccgaca
     3421 acaccacgga attgtcagtg cccaacagcc gagcccctgt ccagcagcgg gcaaggcagg
     3481 cggcgatgag ttccgccgtg gcaataggga gggggaaagc gaaagtcccg gaaaggagct
     3541 gacaggtggt ggcaatgccc caaccagtgg gggttgcgtc agcaaacaca gtgcacacca
     3601 cgccacgttg cctgacaacg ggccacaact cctcataaag agacagcaac caggatttat
     3661 acaaggagga gaaaatgaaa gccatacggg aagcaatagc atgatacaaa ggcattaaag
     3721 cagcgtatcc acatagcgta aaaggagcaa catagttaag aataccagtc aatctttcac
     3781 aaattttgta atccagaggt tgattgtcga cttaacgcgt tcaggcaccg ggcttgcggg
     3841 tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc ggcggtgacg gtgaagccga
     3901 gccgctcgta gaaggggagg ttgcggggcg cggaggtctc caggaaggcg ggcaccccgg
     3961 cgcgctcggc cgcctccact ccggggagca cgacggcgct gcccagaccc ttgccctggt
     4021 ggtcgggcga gactccgacg gtggccagga accacgcggg ctccttgggc cggtgcggcg
     4081 ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga accgctcaac tcggccatgc
     4141 gcgggccgat ctcggcgaac accgcccccg cttcgacgct ctccggcgtg gtccagaccg
     4201 ccaccgcggc gccgtcgtcc gcgacccaca ccttgccgat gtcgagcccg acgcgcgtga
     4261 ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg gtccggatcg acggtgtggc
     4321 gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg tacggccctg gggacgtcgt
     4381 cgcgggtggc gaggcgcacc gtgggcttgt actcggtcat ggtggcggat cccgcgtcac
     4441 gacacctgtg ttctggcggc aaacccgttg cgaaaaagaa cgttcacggc gactactgca
     4501 cttatatacg gttctccccc accctcggga aaaaggcgga gccagtacac gacatcactt
     4561 tcccagttta ccccgcgcca ccttctctag gcaccggttc aattgccgac ccctcccccc
     4621 aacttctcgg ggactgtggg cgatgtgcgc tctgcccact gacgggcacc ggagccactc
     4681 gagccggcgc caaagtggat ctctgctgtc cctgtaataa acccgaaaat tttgaatttt
     4741 tgtaatttgt ttttgtaatt ctttagtttg tatgtctgtt gctattatgt ctactattct
     4801 ttcccctgca ctgtaccccc caatcccccc ttttctttta aaattgtgga tgaatactgc
     4861 catttgtctc aagatctaga attcaaaaaa agcaccgact cggtgccact ttttcaagtt
     4921 gataacggac tagccttatt taaacttgct atgctgtttc cagcatagct cttaaacgtg
     4981 tcctcgccca gcaagcacgg tgtttcgtcc tttccacaag atatataaag ccaagaaatc
     5041 gaaatacttt caagttacgg taagcatatg atagtccatt ttaaaacata attttaaaac
     5101 tgcaaactac ccaagaaatt attactttct acgtcacgta ttttgtacta atatctttgt
     5161 gtttacagtc aaattaattc taattatctc tctaacagcc ttgtatcgta tatgcaaata
     5221 tgaaggaatc atgggaaata ggccctctct gggtcccctc ggggttggga ggtgggtctg
     5281 aaacgataat ggtgaatatc cctgcctaac tctattcact atagaaagta cagcaaaaac
     5341 tattcttaaa cctaccaagc ctcctactat cattatgaat aattttatat accacagcca
     5401 atttgttatg ttaaaccaat tccacaaact tgcccattta tctaattcca ataattcttg
     5461 ttcattcttt tcttgctggt tttgcgattc ttcaattaag gagtgtatta agcttgtgta
     5521 attgttaatt tctctgtccc actccatcca ggtcgtgtga ttccaaatct gttccagaga
     5581 tttattactc caactagcat tccaaggcac agcagtggtg caaatgagtt ttccagagca
     5641 accccaaatc cccaggagct gttgatcctt taggtatctt tccacagcca ggattcttgc
     5701 ctggagctgc ttgatgcccc agactgtgag ttgcaacaga tgctgttgcg cctcaatagc
     5761 cctcagcaaa ttgttctgct gctgcactat accagacaat aattgtctgg cctgtaccgt
     5821 cagcgtcatt gacgctgcgc ccatagtgct tcctgctgct cccaagaacc caaggaacaa
     5881 agctcctatt cccactgctc ttttttctct ctgcaccact cttctctttg ccttggtggg
     5941 tgctactcct aatggttcaa tttttactac tttatattta tataattcac ttctccaatt
     6001 gtccctcata tctcctcctc caggtctgaa gatcagcggc cgcttgctgt gcggtggtct
     6061 tacttttgtt ttgctcttcc tctatcttgt ctaaagcttc cttggtgtct tttatctcta
     6121 tcctttgatg cacacaatag agggttgcta ctgtattata taatgatcta agttcttctg
     6181 atcctgtctg aagggatggt tgtagctgtc ccagtatttg tctacagcct tctgatgttt
     6241 ctaacaggcc aggattaact gcgaatcgtt ctagctccct gcttgcccat actatatgtt
     6301 ttaatttata ttttttcttt ccccctggcc ttaaccgaat tttttcccat cgcgatctaa
     6361 ttctcccccg cttaatactg acgctctcgc acccatctct ctccttctag cctccgctag
     6421 tcaaaatttt tggcgtactc accagtcgcc gcccctcgcc tcttgccgtg cgcgcttcag
     6481 caagccgagt cctgcgtcga gagagctcct ctggtttccc tttcgctttc aagtccctgt
     6541 tcgggcgcca ctgctagaga ttttccacac tgactaaaag ggtctgaggg atctctagtt
     6601 accagagtca cacaacagac gggcacacac tacttgaagc actcaaggca agctttattg
     6661 aggcttaagc agtgggttcc ctagttagcc agagagctcc caggctcaga tctggtctaa
     6721 ccagagagac ccgtttatgt atcgagctag gcacttaaat acaatatctc tgcaatgcgg
     6781 caattcagtg gttcgtccaa tccatgtcag acccgtctgt tgccttccta ataaggcacg
     6841 atcgtaccac cttacttcca ccaatcggca tgcacggtgc tttttctctc cttgtaaggc
     6901 atgttgctaa ctcatcgtta ccatgttgca agactacaag agtattgcat aagactacat
     6961 taagcttgca gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     7021 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     7081 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     7141 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     7201 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     7261 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cg
//
