LOCUS       sequence-183557-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human ACTR1A.
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: ACTR1A C9.5 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    134..155
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(170..175,176..193,194..200)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    208..224
                     /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     213..235
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     232..248
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     232..248
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     267..287
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        269..287
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        315..541
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             542..722
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    769..894
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1387..1620
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1805..1849
                     /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        2027..2275
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2027..2047
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2198..2217
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2431..2548
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2617..2828
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2854..3453
                     /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(2854..2873)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3350..3370
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3469..4057
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3522..3542)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3940..3951)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4129..4362
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4440..4561
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4477..4496)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4531..4550
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4601..4736
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4756..4775)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4757..4775)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4785..4802)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4785..4801)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4794..4816)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4943..5398
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5030..5049)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5240..5261
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5424..5528
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5529..5597,5598..6389)
                     /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(5747..5766)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6560..7148
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7049..7068
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(7302,1..17)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
       61 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
      121 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
      181 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
      241 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
      301 agctgcaagc ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat
      361 gagttagcaa catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt
      421 aaggtggtac gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg
      481 aaccactgaa ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa
      541 cgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
      601 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
      661 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
      721 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
      781 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
      841 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
      901 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
      961 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
     1021 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
     1081 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     1141 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     1201 caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg
     1261 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     1321 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     1381 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     1441 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     1501 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     1561 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     1621 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     1681 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     1741 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     1801 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     1861 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     1921 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     1981 accattatcg tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc
     2041 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa
     2101 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     2161 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     2221 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgggat
     2281 ccggtgtgat taaagcgttt aagagctatg ctggaaacag catagcaagt ttaaataagg
     2341 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg aattctagat
     2401 cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     2461 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     2521 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact
     2581 ttggcgccgg ctcgagtggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
     2641 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
     2701 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
     2761 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
     2821 gaacacaggt gtcgtgacgc gggatccgcc accatgaccg agtacaagcc cacggtgcgc
     2881 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     2941 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     3001 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     3061 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     3121 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     3181 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga
     3241 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     3301 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     3361 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     3421 acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg
     3481 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     3541 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     3601 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     3661 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     3721 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     3781 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     3841 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     3901 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     3961 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     4021 cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt
     4081 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     4141 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     4201 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     4261 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     4321 gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat agtagttcat
     4381 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga
     4441 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4501 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4561 atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc
     4621 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4681 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4741 gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta
     4801 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     4861 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     4921 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4981 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     5041 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5101 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     5161 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5221 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     5281 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     5341 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     5401 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5461 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5521 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5581 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5641 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5701 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5761 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5821 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5881 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5941 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     6001 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6061 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6121 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6181 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6241 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6301 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6361 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6421 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6481 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6541 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6601 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6661 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6721 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6781 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6841 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6901 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6961 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     7021 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7081 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7141 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     7201 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     7261 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg ca
//
