LOCUS       sequence-183153-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human SUGT1.
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: SUGT1 E10.4 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     57..74
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    191..212
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(227..232,233..250,251..257)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    265..281
                     /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     270..292
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     289..305
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     289..305
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     324..344
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        326..344
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        372..598
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             599..779
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    826..951
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1444..1677
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1862..1906
                     /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        2084..2332
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2084..2104
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2255..2274
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2488..2605
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2674..2885
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2911..3510
                     /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(2911..2930)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3407..3427
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3526..4114
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3579..3599)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3997..4008)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4186..4419
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4497..4618
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4534..4553)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4588..4607
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4658..4793
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4813..4832)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4814..4832)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4842..4859)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4842..4858)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4851..4873)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5000..5455
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5087..5106)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5297..5318
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5481..5585
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5586..5654,5655..6446)
                     /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(5804..5823)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6617..7205
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7106..7125
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
       61 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
      121 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
      181 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
      241 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
      301 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc
      361 tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag
      421 ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag
      481 gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac
      541 cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg
      601 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      661 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      721 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
      781 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
      841 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
      901 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
      961 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
     1021 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
     1081 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
     1141 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
     1201 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
     1261 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
     1321 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1381 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1441 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1501 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1561 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1621 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1681 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1741 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     1801 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     1861 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     1921 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     1981 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2041 attatcgttt cagacccacc tcccaacccc gaggggaccc agagagggcc tatttcccat
     2101 gattccttca tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt
     2161 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     2221 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     2281 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgctcggat
     2341 gccctaatcg acggtttaag agctatgctg gaaacagcat agcaagttta aataaggcta
     2401 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttgaat tctagatctt
     2461 gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     2521 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     2581 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg
     2641 gcgccggctc gagtggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc
     2701 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg
     2761 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac
     2821 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
     2881 cacaggtgtc gtgacgcggg atccgccacc atgaccgagt acaagcccac ggtgcgcctc
     2941 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     3001 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     3061 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     3121 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     3181 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     3241 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc
     3301 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     3361 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     3421 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     3481 tggtgcatga cccgcaagcc cggtgcctga acgcgttaag tcgacaatca acctctggat
     3541 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     3601 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     3661 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     3721 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     3781 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     3841 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3901 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     3961 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     4021 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     4081 acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca
     4141 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     4201 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
     4261 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     4321 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     4381 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tacgtatagt agttcatgtc
     4441 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact
     4501 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     4561 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     4621 atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4681 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4741 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac
     4801 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     4861 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     4921 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     4981 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     5041 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     5101 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     5161 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     5221 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     5281 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     5341 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     5401 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     5461 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5521 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5581 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5641 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5701 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5761 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5821 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5881 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5941 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6001 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6061 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6121 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6181 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6241 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6301 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6361 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6421 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6481 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6541 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6601 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6661 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6721 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6781 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6841 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6901 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6961 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     7021 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     7081 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     7141 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7201 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7261 acatgttctt tcctgcgtta tcccctgatt ctgtggataa cc
//
