LOCUS       sequence-183028-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human MAD1L1.
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: MAD1L1 E10.1 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     90..107
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    224..245
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(260..265,266..283,284..290)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    298..314
                     /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     303..325
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     322..338
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     322..338
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     357..377
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        359..377
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        405..631
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             632..812
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    859..984
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1477..1710
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1895..1939
                     /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        2117..2365
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2117..2137
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2288..2307
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2521..2638
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2707..2918
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2944..3543
                     /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(2944..2963)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3440..3460
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3559..4147
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3612..3632)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4030..4041)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4219..4452
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4530..4651
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4567..4586)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4621..4640
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4691..4826
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4846..4865)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4847..4865)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4875..4892)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4875..4891)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4884..4906)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5033..5488
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5120..5139)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5330..5351
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5514..5618
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5619..5687,5688..6479)
                     /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(5837..5856)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6650..7238
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7139..7158
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
       61 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
      121 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
      181 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
      241 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
      301 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa
      361 ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt cttatgcaat
      421 actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac aaggagagaa
      481 aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct tattaggaag
      541 gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt gcagagatat
      601 tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc agatctgagc
      661 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
      721 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
      781 acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg
      841 aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca
      901 agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag
      961 gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa
     1021 aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca
     1081 agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt
     1141 agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca
     1201 ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc
     1261 aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa
     1321 gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt
     1381 atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag
     1441 aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt
     1501 gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca
     1561 attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca
     1621 gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt
     1681 ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat
     1741 ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg
     1801 gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca
     1861 ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt
     1921 agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa
     1981 attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc
     2041 tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc acctcccaac
     2101 cccgagggga cccagagagg gcctatttcc catgattcct tcatatttgc atatacgata
     2161 caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca
     2221 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     2281 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     2341 tatcttgtgg aaaggacgaa acaccgcaac ttcatctctc agcgtggttt aagagctatg
     2401 ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga aaaagtggca
     2461 ccgagtcggt gctttttttg aattctagat cttgagacaa atggcagtat tcatccacaa
     2521 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
     2581 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
     2641 ggtttattac agggacagca gagatccact ttggcgccgg ctcgagtggc tccggtgccc
     2701 gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
     2761 attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
     2821 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
     2881 acgttctttt tcgcaacggg tttgccgcca gaacacaggt gtcgtgacgc gggatccgcc
     2941 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     3001 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     3061 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     3121 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     3181 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     3241 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     3301 gagcccgcgt ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg
     3361 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     3421 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     3481 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     3541 tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     3601 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     3661 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     3721 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     3781 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3841 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3901 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     3961 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     4021 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     4081 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     4141 tccccgcgtc gactttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
     4201 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt
     4261 ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     4321 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     4381 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     4441 aaatctctag cagtacgtat agtagttcat gtcatcttat tattcagtat ttataacttg
     4501 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca
     4561 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4621 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct
     4681 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4741 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     4801 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg
     4861 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     4921 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     4981 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc
     5041 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5101 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5161 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5221 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     5281 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5341 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     5401 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5461 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     5521 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5581 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5641 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5701 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5761 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5821 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5881 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5941 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6001 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6061 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6121 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6181 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6241 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6301 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6361 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6421 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6481 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6541 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6601 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6661 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6721 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6781 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6841 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6901 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6961 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7021 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7081 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7141 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7201 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7261 tacggttcct ggccttttgc tggccttttg ctcacatgtt ct
//
