LOCUS       sequence-432525-        3933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A piggyBac transposon-based gRNA expression vector, to allow for 
            genomic integration and stable expression of gRNAs. Contains both 
            antibiotic (puromycin) and fluorophore (GFP) markers..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3933)
  AUTHORS   Chardon FM, McDiarmid TA, Page NF, Daza RM, Martin BK, Domcke S, 
            Regalado SG, Lalanne JB, Calderon D, Li X, Starita LM, Sanders SJ, 
            Ahituv N, Shendure J
  TITLE     Multiplex, single-cell CRISPRa screening for cell type specific 
            regulatory elements.
  JOURNAL   Nat Commun. 2024 Sep 18;15(1):8209. doi: 10.1038/s41467-024-52490-4.
  PUBMED    39294132
REFERENCE   2  (bases 1 to 3933)
  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"; journalName: "Nat 
            Commun."; date: "2024-09-18"; volume: "15(1):8209. doi"; pages: " 
            10.1038/s41467-024-52490-4"
COMMENT     Sequence Label: piggyFlex
FEATURES             Location/Qualifiers
     source          1..3933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   275..309
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(800..816)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(800..816)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(813..835)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    824..840
                     /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)."
     promoter        complement(join(848..854,855..872,873..878))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    893..914
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1031..1048)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1202..1790)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1282..1301)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1961..2752,2753..2821))
                     /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     2584..2603
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2822..2926)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2952..3407)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3089..3110)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3301..3320
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3534..3556
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3548..3565
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3549..3565
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   3695..3757
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata
       61 acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca
      121 ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac
      181 gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg
      241 ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc
      301 tttctagggt taatctagct gcatcaggat catatcgtcg ggtctttttt ccggctcagt
      361 catcgcccaa gctggcgcta tctgggcatc ggggaggaag aagcccgtgc cttttcccgc
      421 gaggttgaag cggcatggaa agagtttgcc gaggatgact gctgctgcat tgacgttgag
      481 cgaaaacgca cgtttaccat gatgattcgg gaaggtgtgg ccatgcacgc ctttaacggt
      541 gaactgttcg ttcaggccac ctgggatacc agttcgtcgc ggcttttccg gacacagttc
      601 cggatggtca gcccgaagcg catcagcaac ccgaacaata ccggcgacag ccggaactgc
      661 cgtgccggtg tgcagattaa tgacagcggt gcggcgctgg gatattacgt cagcgaggac
      721 gggtatcctg gctggatgcc gcagaaatgg acatggatac cccgtgagtt acccggcggg
      781 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
      841 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
      901 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
      961 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgca
     1021 gatccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     1081 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     1141 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     1201 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     1261 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     1321 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     1381 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     1441 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     1501 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     1561 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     1621 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     1681 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     1741 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     1801 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     1861 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     1921 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     1981 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     2041 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     2101 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     2161 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     2221 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     2281 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     2341 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     2401 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     2461 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     2521 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     2581 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     2641 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     2701 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     2761 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     2821 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     2881 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     2941 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     3001 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     3061 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     3121 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     3181 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     3241 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     3301 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     3361 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt
     3421 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     3481 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     3541 acgacgttgt aaaacgacgg ccagtgagcg cgcctcgttc attcacgttt ttgaacccgt
     3601 ggaggacggg cagactcgcg gtgcaaatgt gttttacagc gtgatggagc agatgaagat
     3661 gctcgacacg ctgcagaaca cgcagctaga ttaaccctag aaagataatc atattgtgac
     3721 gtacgttaaa gataatcatg tgtaaaattg acgcatgtgt tttatcggtc tgtatatcga
     3781 ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt acatactaat
     3841 aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa caaaaactca
     3901 aaatttcttc tataaagtaa caaaactttt atg
//
