LOCUS       sequence-183811-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human NIN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7301)
  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 7301)
  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: NIN G11.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    59..80
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(95..100,101..118,119..125)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    133..149
                     /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     138..160
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     157..173
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     157..173
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     192..212
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        194..212
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        240..466
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             467..647
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    694..819
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1312..1545
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1730..1774
                     /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        1952..2200
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1952..1972
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2123..2142
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2355..2472
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2541..2752
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2778..3377
                     /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(2778..2797)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3274..3294
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3393..3981
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3446..3466)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3864..3875)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4053..4286
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4364..4485
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4401..4420)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4455..4474
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4525..4660
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4680..4699)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4681..4699)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4709..4726)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4709..4725)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4718..4740)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4867..5322
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4954..4973)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5164..5185
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5348..5452
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5453..5521,5522..6313)
                     /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(5671..5690)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6484..7072
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6973..6992
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7226..7243
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
       61 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
      121 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
      181 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat
      241 gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc
      301 cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg
      361 tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc
      421 gcattgcaga gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt
      481 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
      541 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
      601 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
      661 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
      721 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
      781 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
      841 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
      901 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
      961 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
     1021 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
     1081 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
     1141 accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa
     1201 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
     1261 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
     1321 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
     1381 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
     1441 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
     1501 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
     1561 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
     1621 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
     1681 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
     1741 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
     1801 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
     1861 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
     1921 gacccacctc ccaaccccga ggggacccag agagggccta tttcccatga ttccttcata
     1981 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
     2041 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     2101 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     2161 tttcttggct ttatatatct tgtggaaagg acgaaacacc gtgggaagcg ttacggacga
     2221 gtttaagagc tatgctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac
     2281 ttgaaaaagt ggcaccgagt cggtgctttt tttgaattct agatcttgag acaaatggca
     2341 gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga
     2401 atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa
     2461 attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcg ccggctcgag
     2521 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
     2581 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
     2641 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
     2701 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg
     2761 acgcgggatc cgccaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     2821 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     2881 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     2941 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     3001 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     3061 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     3121 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc
     3181 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     3241 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     3301 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     3361 gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg
     3421 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     3481 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     3541 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     3601 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     3661 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     3721 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     3781 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     3841 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     3901 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     3961 ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga
     4021 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     4081 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
     4141 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     4201 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     4261 ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca
     4321 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc
     4381 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4441 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta
     4501 gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4561 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     4621 gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc
     4681 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     4741 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     4801 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     4861 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4921 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4981 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5041 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     5101 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5161 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     5221 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     5281 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     5341 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5401 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5461 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5521 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5581 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5641 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5701 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5761 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5821 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5881 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5941 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6001 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6061 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6121 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6181 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6241 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6301 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6361 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6421 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6481 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6541 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6601 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6661 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6721 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6781 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6841 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6901 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6961 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7021 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7081 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     7141 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     7201 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     7261 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat t
//
