LOCUS       sequence-183340-        7301 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human CTPS1.
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: CTPS1 E12.3 gRNA
FEATURES             Location/Qualifiers
     source          1..7301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    68..89
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(104..109,110..127,128..134)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    142..158
                     /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     147..169
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     166..182
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     166..182
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     201..221
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        203..221
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        249..475
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             476..656
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    703..828
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1321..1554
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1739..1783
                     /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        1961..2209
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1961..1981
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2132..2151
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2364..2481
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2550..2761
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2787..3386
                     /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(2787..2806)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3283..3303
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3402..3990
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3455..3475)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3873..3884)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4062..4295
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4373..4494
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4410..4429)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4464..4483
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4534..4669
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4689..4708)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4690..4708)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4718..4735)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4718..4734)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4727..4749)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4876..5331
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4963..4982)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5173..5194
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5357..5461
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5462..5530,5531..6322)
                     /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(5680..5699)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6493..7081
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6982..7001
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7235..7252
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
       61 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      121 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
      181 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc
      241 aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
      301 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
      361 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
      421 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc
      481 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      541 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      601 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      661 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
      721 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
      781 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
      841 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
      901 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
      961 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     1021 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     1081 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     1141 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     1201 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     1261 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     1321 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     1381 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     1441 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     1501 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     1561 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     1621 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     1681 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     1741 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat
     1801 aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt
     1861 aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt
     1921 atcgtttcag acccacctcc caaccccgag gggacccaga gagggcctat ttcccatgat
     1981 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac
     2041 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     2101 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     2161 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg agtattgtgc
     2221 ccacactgcg tttaagagct atgctggaaa cagcatagca agtttaaata aggctagtcc
     2281 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgaattcta gatcttgaga
     2341 caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca
     2401 ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa
     2461 attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggcgc
     2521 cggctcgagt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
     2581 gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa
     2641 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
     2701 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
     2761 ggtgtcgtga cgcgggatcc gccaccatga ccgagtacaa gcccacggtg cgcctcgcca
     2821 cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     2881 ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     2941 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     3001 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     3061 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     3121 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc
     3181 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     3241 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     3301 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     3361 gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct ctggattaca
     3421 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3481 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3541 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3601 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3661 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3721 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3781 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     3841 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     3901 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     3961 gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc
     4021 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     4081 ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg
     4141 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     4201 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     4261 cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt catgtcatct
     4321 tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt
     4381 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4441 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4501 ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4561 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4621 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac
     4681 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     4741 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     4801 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     4861 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4921 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4981 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5041 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5101 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5161 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5221 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5281 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     5341 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5401 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5461 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5521 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5581 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5641 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5701 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5761 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5821 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5881 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5941 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6001 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6061 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6121 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6181 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6241 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6301 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6361 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6421 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6481 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6541 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6601 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6661 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6721 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6781 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6841 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6901 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6961 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7021 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7081 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7141 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     7201 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     7261 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg c
//
