LOCUS       sequence-323561-        6582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for pooled cloning of Perturb-seq libraries.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6582)
  AUTHORS   Dixit A, Parnas O, Li B, Chen J, Fulco CP, Jerby-Arnon L, Marjanovic
            ND, Dionne D, Burks T, Raychowdhury R, Adamson B, Norman TM, Lander 
            ES, Weissman JS, Friedman N, Regev A
  TITLE     Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell
            RNA Profiling of Pooled Genetic Screens.
  JOURNAL   Cell. 2016 Dec 15;167(7):1853-1866.e17. doi: 
            10.1016/j.cell.2016.11.038.
  PUBMED    27984732
REFERENCE   2  (bases 1 to 6582)
  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.cell.2016.11"; journalName: "Cell"; date: "2016-12-15- 
            15"; volume: "167"; issue: "7"; pages: "1853-1866.e17"
COMMENT     Sequence Label: pPS
FEATURES             Location/Qualifiers
     source          1..6582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(200..304)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(379..398)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        570..949
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        951..1149
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1104..1124
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             1167..1347
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1394..1519
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2016..2249
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2434..2478
                     /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"
     misc_feature    2776..2893
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     polyA_signal    2952..3176
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3165..3182
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(3285..3307)
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     promoter        complement(3449..3697)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3507..3526)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3677..3697)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(3714..3747)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3803..4391
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3856..3876)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4274..4285)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4394..4410)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4395..4411)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4920..5100
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5162..5750)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5242..5261)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5921..6582,1..130,131..199))
                     /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     6544..6563
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
       61 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
      121 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
      181 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
      241 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
      301 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
      361 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
      421 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
      481 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
      541 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      601 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      661 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      721 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      781 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      841 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      901 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      961 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     1021 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     1081 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     1141 tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg
     1201 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     1261 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     1321 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
     1381 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
     1441 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
     1501 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
     1561 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
     1621 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
     1681 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
     1741 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
     1801 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
     1861 cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta
     1921 tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga
     1981 agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg
     2041 ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa
     2101 ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag
     2161 catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg
     2221 gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt
     2281 tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg
     2341 aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac
     2401 tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta
     2461 gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa
     2521 ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct
     2581 atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc
     2641 ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag agacagagac
     2701 agatccattc gattagtgaa cggatcggca ctgcgtgcgc caattctgca gacaaatggc
     2761 agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag
     2821 aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa
     2881 aattcaaaat tttcgggttt attacaggga cagcagagat ccagtttggt tagtaccggg
     2941 cccgctctag accatagagc ccaccgcatc cccagcatgc ctgctattgt cttcccaatc
     3001 ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca
     3061 atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt
     3121 caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga
     3181 ggctgatcag cgggtttaaa cgggccctct aggaattcat gcatgcgaat tcaattcgcc
     3241 agggttttcc cagtcacgac gcggccgcat gcatgcgcgg ccgccaccaa acgtggcttg
     3301 ccagcccaaa aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta
     3361 tttaaacttg ctatgctgtt tccagcatag ctcttaaaca gagacgttga agatgaaacg
     3421 tgtagtgtgc tcatttacaa ccgtctccgg tgtttcgtcc tttccacaag atatataaag
     3481 ccaagaaatc gaaatacttt caagttacgg taagcatatg atagtccatt ttaaaacata
     3541 attttaaaac tgcaaactac ccaagaaatt attactttct acgtcacgta ttttgtacta
     3601 atatctttgt gtttacagtc aaattaattc taattatctc tctaacagcc ttgtatcgta
     3661 tatgcaaata tgaaggaatc atgggaaata ggccctcaac gtcgagggac ctaataactt
     3721 cgtatagcat acattatacg aagttataca tgtttaaggg ttccggttcc actaggtaca
     3781 attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac
     3841 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     3901 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     3961 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     4021 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     4081 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     4141 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     4201 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     4261 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     4321 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     4381 cgcctccccg catcgatacc gtcgacctcg atcgagacct agaaaaacat ggagcaatca
     4441 caagtagcaa tacagcagct accaatgctg attgtgcctg gctagaagca caagaggagg
     4501 aggaggtggg ttttccagtc acacctcagg tacctttaag accaatgact tacaaggcag
     4561 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     4621 aacgaagaca agatatcctt gatctgtgga tctaccacac acaaggctac ttccctgatt
     4681 ggcagaacta cacaccaggg ccagggatca gatatccact gacctttgga tggtgctaca
     4741 agctagtacc agttgagcaa gagaaggtag aagaagccaa tgaaggagag aacacccgct
     4801 tgttacaccc tgtgagcctg catgggatgg atgacccgga gagagaagta ttagagtgga
     4861 ggtttgacag ccgcctagca tttcatcaca tggcccgaga gctgcatccg gactgtactg
     4921 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     4981 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     5041 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     5101 gcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5161 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5221 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5281 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5341 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5401 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5461 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5521 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5581 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5641 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5701 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5761 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5821 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5881 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5941 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6001 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6061 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6121 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6181 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6241 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6301 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6361 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6421 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6481 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6541 gggataatac cgcgccacat agcagaactt taaaagtgct ca
//
