LOCUS       sequence-183452-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human AHCTF1.
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: AHCTF1 A7.2 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..30
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    147..168
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(183..188,189..206,207..213)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    221..237
                     /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     226..248
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     245..261
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     245..261
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     280..300
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        282..300
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        328..554
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             555..735
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    782..907
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1400..1633
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1818..1862
                     /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        2040..2288
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2040..2060
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2211..2230
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2630..2841
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2867..3466
                     /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(2867..2886)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3363..3383
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3482..4070
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3535..3555)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3953..3964)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4142..4375
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4453..4574
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4490..4509)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4544..4563
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4614..4749
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4769..4788)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4770..4788)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4798..4815)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4798..4814)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4807..4829)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4956..5411
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5043..5062)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5253..5274
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5437..5541
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5542..5610,5611..6402)
                     /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(5760..5779)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6573..7161
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7062..7081
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
       61 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
      121 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
      181 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
      241 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
      301 gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca
      361 acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc
      421 gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga
      481 catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag
      541 ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct
      601 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
      661 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
      721 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
      781 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
      841 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
      901 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
      961 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
     1021 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
     1081 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
     1141 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
     1201 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca
     1261 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
     1321 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
     1381 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
     1441 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
     1501 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
     1561 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
     1621 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
     1681 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
     1741 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
     1801 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
     1861 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag
     1921 taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta
     1981 ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccagag
     2041 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     2101 taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     2161 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     2221 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     2281 gaaacaccga gggcttgaag agtcacttcg tttaagagct atgctggaaa cagcatagca
     2341 agtttaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     2401 ttgaattcta gatcttgaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc actttggcgc cggctcgagt ggctccggtg cccgtcagtg ggcagagcgc
     2641 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag
     2701 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc
     2761 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac
     2821 gggtttgccg ccagaacaca ggtgtcgtga cgcgggatcc gccaccatga ccgagtacaa
     2881 gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc
     2941 cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg
     3001 ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg
     3061 ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg
     3121 ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc
     3181 gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct
     3241 ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct
     3301 ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc
     3361 ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc
     3421 cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga
     3481 caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     3541 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     3601 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     3661 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     3721 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     3781 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     3841 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     3901 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     3961 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     4021 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta
     4081 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     4141 ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct
     4201 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     4261 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     4321 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg
     4381 tatagtagtt catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca
     4441 gagagtgaga ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4501 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4561 atcaatgtat cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc
     4621 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4681 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     4741 ttggaggcct agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     4801 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     4861 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     4921 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag
     4981 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     5041 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5101 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5161 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5221 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5281 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     5341 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     5401 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5461 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5521 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5581 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5641 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5701 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5761 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5821 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5881 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5941 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     6001 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6061 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     6121 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6181 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6241 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6301 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6361 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6421 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6481 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6541 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6601 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6661 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6721 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6781 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6841 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6901 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6961 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     7021 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     7081 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7141 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     7201 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     7261 attaccgcct ttgagtgagc tgataccgct cgccgcagcc ga
//
