LOCUS       sequence-169783-        3936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac vector for expressing modified sgRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3936)
  AUTHORS   Seirin-Lee S, Osakada F, Takeda J, Tashiro S, Kobayashi R, Yamamoto 
            T, Ochiai H
  TITLE     Role of dynamic nuclear deformation on genomic architecture 
            reorganization.
  JOURNAL   PLoS Comput Biol. 2019 Sep 11;15(9):e1007289. doi: 
            10.1371/journal.pcbi.1007289. eCollection 2019 Sep.
  PUBMED    31509522
REFERENCE   2  (bases 1 to 3936)
  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: "PLoS Comput
            Biol."; date: "2019-09-11"; volume: "15(9):e1007289. doi"; pages: " 
            10.1371/journal.pcbi.1007289. eCollection 2019 Sep"
COMMENT     Sequence Label: pLR5-U6-sgRNA-EF
FEATURES             Location/Qualifiers
     source          1..3936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    31..52
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(67..72,73..90,91..97)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    105..121
                     /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     110..132
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     129..145
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     129..145
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     164..184
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        166..184
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   235..297
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(673..921)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(731..750)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(901..921)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   1224..1258
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1287..1306)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1288..1306)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1316..1333)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1316..1332)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1325..1347)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1474..1929
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1561..1580)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1771..1792
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1955..2059
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2060..2128,2129..2920)
                     /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(2278..2297)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3091..3679
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3580..3599
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3833..3850
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
       61 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
      121 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
      181 aagggaacaa aagctggatt aattaagcgg ccgcatacgc gtatactaga ttaaccctag
      241 aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt
      301 tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa gttttattat
      361 atttacactt acatactaat aataaattca acaaacaatt tatttatgtt tatttattta
      421 ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt taactagtga
      481 gacggccatt tgtctgcaga attggcgcac gcgctaaaaa cggactagcc ttattttaac
      541 ttgctatttc tagctctaaa acaaaaaagc accgactcgg tgccactttt tcaagttgat
      601 aacggactag ccttatttaa acttgctatg ctgtttccag catagctctt aaacaggtct
      661 tctcgaagac ccggtgtttc gtcctttcca caagatatat aaagccaaga aatcgaaata
      721 ctttcaagtt acggtaagca tatgatagtc cattttaaaa cataatttta aaactgcaaa
      781 ctacccaaga aattattact ttctacgtca cgtattttgt actaatatct ttgtgtttac
      841 agtcaaatta attccaatta tctctctaac agccttgtat cgtatatgca aatatgaagg
      901 aatcatggga aataggccct ccatgcgaga cggtgctagc attctagttg atatctataa
      961 caagaaaata tatatataat aagttatcac gtaagtagaa catgaaataa caatataatt
     1021 atcgtatgag ttaaatctta aaagtcacgt aaaagataat catgcgtcat tttgactcac
     1081 gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg cctcacggga
     1141 gctccaagcg gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa
     1201 gagagagcaa tatttcaaga atgcatgcgt caattttacg cagactatct ttctagggtt
     1261 aatctagaat ggcgcgcccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     1321 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     1381 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     1441 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     1501 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1561 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1621 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1681 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1741 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1801 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1861 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1921 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1981 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     2041 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     2101 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     2161 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     2221 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     2281 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     2341 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     2401 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     2461 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     2521 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     2581 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     2641 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     2701 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     2761 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     2821 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     2881 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     2941 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     3001 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3061 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3121 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3181 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     3241 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3301 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3361 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3421 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3481 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3541 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3601 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3661 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3721 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3781 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3841 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     3901 gattcattaa tgcagctggc acgacaggtt tcccga
//
