LOCUS       sequence-473287-        5619 bp    DNA     circular SYN 20-MAY-2025
DEFINITION  Blank donor for Perturb Seq landing pad with Pa01 recombinase at the
            AAVS1 loci.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5619)
  AUTHORS   Sivakumar S, Wang Y, Goetsch SC, Pandit V, Wang L, Zhao H, 
            Sundarrajan A, Armendariz D, Takeuchi C, Nzima M, Chen WC, Dederich 
            AE, El Hayek L, Gao T, Ghazawi R, Gogate A, Kaur K, Kim HB, McCoy 
            MK, Niederstrasser H, Oura S, Pinzon-Arteaga CA, Sanghvi M, Schmitz 
            DA, Yu L, Zhang Y, Zhou Q, Kraus WL, Xu L, Wu J, Posner BA, Chahrour
            MH, Hon GC, Munshi NV
  TITLE     Benchmarking and optimizing Perturb-seq in differentiating human 
            pluripotent stem cells.
  JOURNAL   bioRxiv [Preprint]. 2025 Jan 23:2025.01.21.633969. doi: 
            10.1101/2025.01.21.633969.
  PUBMED    39896670
REFERENCE   2  (bases 1 to 5619)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 20, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 Jan 23:2025.01.21.633969. doi: 
            10.1101/2025.01.21.633969."
COMMENT     Sequence Label: Donor-attp-u6-blank-sgrna-backbone
FEATURES             Location/Qualifiers
     source          1..5619
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(242..1033,1034..1102))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     polyA_signal    1207..1255
                     /note="synthetic polyadenylation signal"
     misc_feature    1269..1360
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     1309..1328
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        1381..2559
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1612..2550
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2507..2527
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     LTR             2687..2867
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2914..3039
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3532..3765
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3950..3994
                     /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        4172..4420
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4172..4192
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4343..4362
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    4608..4725
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     complement(4902..4919)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5073..5619,1..42))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5153..5172)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
       61 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
      121 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
      181 taaagtatat atgagtaaac ttggtctgac agcggccgca aatgctaaac cactgcagtg
      241 gttaccagtg cttgatcagt gaggcaccga tctcagcgat ctgcctattt cgttcgtcca
      301 tagtggcctg actccccgtc gtgtagatca ctacgattcg tgagggctta ccatcaggcc
      361 ccagcgcagc aatgatgccg cgagagccgc gttcaccggc ccccgatttg tcagcaatga
      421 accagccagc agggagggcc gagcgaagaa gtggtcctgc tactttgtcc gcctccatcc
      481 agtctatgag ctgctgtcgt gatgctagag taagaagttc gccagtgagt agtttccgaa
      541 gagttgtggc cattgctact ggcatcgtgg tatcacgctc gtcgttcggt atggcttcgt
      601 tcaactctgg ttcccagcgg tcaagccggg tcacatgatc acccatatta tgaagaaatg
      661 cagtcagctc cttagggcct ccgatcgttg tcagaagtaa gttggccgcg gtgttgtcgc
      721 tcatggtaat ggcagcacta cacaattctc ttaccgtcat gccatccgta agatgctttt
      781 ccgtgaccgg cgagtactca accaagtcgt tttgtgagta gtgtatacgg cgaccaagct
      841 gctcttgccc ggcgtctata cgggacaaca ccgcgccaca tagcagtact ttgaaagtgc
      901 tcatcatcgg gaatcgttct tcggggcgga aagactcaag gatcttgccg ctattgagat
      961 ccagttcgat atagcccact cttgcaccca gttgatcttc agcatctttt actttcacca
     1021 gcgtttcggg gtgtgcaaaa acaggcaagc aaaatgccgc aaagaaggga atgagtgcga
     1081 cacgaaaatg ttggatgctc atactcgtcc tttttcaata ttattgaagc atttatcagg
     1141 gttactagta cgtctgtcaa ggataagtaa gtaatattaa ggtacgggag gtattggaca
     1201 ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgaatcg
     1261 atagtactaa catacgctct ccatcaaaac aaaacgaaac aaaacaaact agcaaaatag
     1321 gctgtcccca gtgcaagtgc aggtgccaga acatttctct tcttgaaagg agtgggaatt
     1381 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
     1441 ggaggggtcg gcaattgatc cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
     1501 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
     1561 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
     1621 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
     1681 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
     1741 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
     1801 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
     1861 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
     1921 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
     1981 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
     2041 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
     2101 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     2161 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     2221 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     2281 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     2341 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     2401 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     2461 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     2521 gtggttcaaa gtttttttct tccatttcag gtgtcgtgat ctctgtaacg ctcttcgaga
     2581 aagcagattc tcatatccat cttgagtctt ctttctcgca agacaacacg aaatagacac
     2641 agtctcttcc ctagctgtac actgagccgt gctttttgct tgtactgggt ctctctggtt
     2701 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     2761 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     2821 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
     2881 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
     2941 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
     3001 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
     3061 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
     3121 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
     3181 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
     3241 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
     3301 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
     3361 accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa
     3421 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
     3481 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
     3541 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
     3601 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
     3661 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
     3721 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
     3781 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
     3841 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
     3901 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
     3961 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
     4021 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
     4081 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
     4141 gacccacctc ccaaccccga ggggacccag agagggccta tttcccatga ttccttcata
     4201 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
     4261 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     4321 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     4381 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggagacggga taccgtctct
     4441 gtttaagagc taagctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac
     4501 ttggccgctt taaggccggt cctagcaagg ccaagtggca ccgagtcggt gcttttttaa
     4561 gcttggcgta actagatctt gagacaaatg gcagtattca tccacaattt taaaagaaaa
     4621 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     4681 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     4741 gacagaaatc gataaggatc cgtcgaccga tgcccttgag agccttcaac ccagtcagct
     4801 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca
     4861 tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt cctcgctcac tgactcgctg
     4921 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4981 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5041 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5101 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5161 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5221 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5281 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5341 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5401 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5461 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     5521 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5581 tccggcaaac aaaccaccgc tggtagcggt ggttttttt
//
