LOCUS       sequence-182569-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human GINS1.
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: GINS1 D11.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    33..54
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(69..74,75..92,93..99)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    107..123
                     /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     112..134
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     131..147
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     131..147
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     166..186
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        168..186
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        214..440
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             441..621
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    668..793
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1286..1519
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1704..1748
                     /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        1926..2174
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1926..1946
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2097..2116
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2330..2447
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2516..2727
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2753..3352
                     /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(2753..2772)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3249..3269
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3368..3956
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3421..3441)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3839..3850)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4028..4261
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4339..4460
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4376..4395)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4430..4449
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4500..4635
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4655..4674)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4656..4674)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4684..4701)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4684..4700)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4693..4715)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4842..5297
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4929..4948)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5139..5160
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5323..5427
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5428..5496,5497..6288)
                     /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(5646..5665)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6459..7047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6948..6967
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7201..7218
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
       61 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
      121 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
      181 taaagggaac aaaagctgga gctgcaagct taatgtagtc ttatgcaata ctcttgtagt
      241 cttgcaacat ggtaacgatg agttagcaac atgccttaca aggagagaaa aagcaccgtg
      301 catgccgatt ggtggaagta aggtggtacg atcgtgcctt attaggaagg caacagacgg
      361 gtctgacatg gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt
      421 gcctagctcg atacataaac gggtctctct ggttagacca gatctgagcc tgggagctct
      481 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
      541 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
      601 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
      661 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
      721 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
      781 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
      841 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
      901 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
      961 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     1021 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     1081 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccgctga
     1141 tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata
     1201 aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc
     1261 agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag
     1321 gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg
     1381 gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc
     1441 aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc
     1501 taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg
     1561 ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga
     1621 cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg
     1681 aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg
     1741 caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa
     1801 tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata
     1861 gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac
     1921 ccagagaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     1981 gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     2041 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     2101 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     2161 aaggacgaaa caccgagctg catcgcgcgc ccgaagttta agagctatgc tggaaacagc
     2221 atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     2281 ctttttttga attctagatc ttgagacaaa tggcagtatt catccacaat tttaaaagaa
     2341 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
     2401 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
     2461 gggacagcag agatccactt tggcgccggc tcgagtggct ccggtgcccg tcagtgggca
     2521 gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt
     2581 gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
     2641 tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt
     2701 cgcaacgggt ttgccgccag aacacaggtg tcgtgacgcg ggatccgcca ccatgaccga
     2761 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     2821 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     2881 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     2941 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     3001 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     3061 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     3121 gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     3181 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     3241 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     3301 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta
     3361 agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     3421 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     3481 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     3541 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     3601 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     3661 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     3721 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     3781 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3841 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     3901 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg
     3961 actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     4021 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact
     4081 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     4141 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     4201 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     4261 agtacgtata gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga
     4321 atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4381 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4441 aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca
     4501 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4561 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     4621 gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg
     4681 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     4741 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     4801 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     4861 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4921 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4981 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5041 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5101 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5161 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     5221 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5281 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5341 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5401 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5461 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5521 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5581 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5641 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5701 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5761 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5821 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5881 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5941 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6001 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6061 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6121 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6181 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6241 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6301 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6361 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6421 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6481 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6541 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6601 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6661 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6721 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6781 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6841 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6901 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6961 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7021 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7081 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     7141 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     7201 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     7261 cgttggccga ttcattaatg cagctggcac gacaggtttc cc
//
