LOCUS       sequence-183288-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human PAFAH1B1.
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: PAFAH1B1 G9.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..22
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    139..160
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(175..180,181..198,199..205)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    213..229
                     /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)."
     primer_bind     218..240
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     237..253
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     237..253
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     272..292
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        274..292
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        320..546
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             547..727
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    774..899
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1392..1625
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1810..1854
                     /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"
     promoter        2032..2280
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2032..2052
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2203..2222
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2436..2553
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2622..2833
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2859..3458
                     /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(2859..2878)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3355..3375
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3474..4062
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3527..3547)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3945..3956)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4134..4367
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4445..4566
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4482..4501)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4536..4555
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4606..4741
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4761..4780)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4762..4780)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4790..4807)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4790..4806)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4799..4821)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4948..5403
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5035..5054)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5245..5266
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5429..5533
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5534..5602,5603..6394)
                     /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     complement(5752..5771)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6565..7153
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7054..7073
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
       61 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
      121 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
      181 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
      241 aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa
      301 gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg caacatggta
      361 acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg ccgattggtg
      421 gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct gacatggatt
      481 ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct agctcgatac
      541 ataaacgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
      601 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
      661 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
      721 tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc
      781 gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga
      841 gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca
      901 gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa
      961 agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag
     1021 ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac
     1081 catcccttca gacaggatca gaagaactta gatcattata taatacagta gcaaccctct
     1141 attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg
     1201 aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt cagacctgga
     1261 ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt
     1321 gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga
     1381 gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc
     1441 gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag
     1501 cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg
     1561 ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag
     1621 ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat
     1681 gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg
     1741 gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac
     1801 cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat
     1861 tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc
     1921 ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga
     1981 tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccag agagggccta
     2041 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga
     2101 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
     2161 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
     2221 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc
     2281 gcgtccgcca gaaaaatatg cgtttaagag ctatgctgga aacagcatag caagtttaaa
     2341 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttgaattc
     2401 tagatcttga gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
     2461 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     2521 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
     2581 ccactttggc gccggctcga gtggctccgg tgcccgtcag tgggcagagc gcacatcgcc
     2641 cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg
     2701 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg
     2761 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc
     2821 cgccagaaca caggtgtcgt gacgcgggat ccgccaccat gaccgagtac aagcccacgg
     2881 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     2941 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     3001 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     3061 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     3121 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     3181 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     3241 tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     3301 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     3361 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     3421 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac
     3481 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     3541 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     3601 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     3661 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     3721 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     3781 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     3841 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg
     3901 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     3961 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     4021 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat
     4081 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
     4141 gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt
     4201 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     4261 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     4321 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta cgtatagtag
     4381 ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga
     4441 gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4501 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4561 atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact
     4621 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4681 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4741 ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     4801 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     4861 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     4921 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     4981 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     5041 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     5101 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     5161 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5221 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     5281 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     5341 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     5401 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5461 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5521 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5581 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5641 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5701 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5761 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5821 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5881 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5941 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     6001 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6061 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6121 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6181 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6241 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6301 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6361 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6421 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6481 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6541 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     6601 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     6661 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     6721 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     6781 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     6841 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6901 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6961 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     7021 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     7081 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     7141 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     7201 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     7261 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc ga
//
