LOCUS       sequence-361257-        7329 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBAC-based sgRNA entry (Esp3I) and Tet-On 3G transactivator 
            expression vector. Insert protospacer and transfect with CRISPRa 
            (183409) or CRISPRi (183410) plasmid for inducible epigenome 
            editing..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7329)
  AUTHORS   Tang WWC, Castillo-Venzor A, Gruhn WH, Kobayashi T, Penfold CA, 
            Morgan MD, Sun D, Irie N, Surani MA
  TITLE     Sequential enhancer state remodelling defines human germline 
            competence and specification.
  JOURNAL   Nat Cell Biol. 2022 Apr;24(4):448-460. doi: 
            10.1038/s41556-022-00878-z. Epub 2022 Apr 11.
  PUBMED    35411086
REFERENCE   2  (bases 1 to 7329)
  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: 
            "10.1038/s41556-022-00878-z"; journalName: "Nat Cell Biol"; date: 
            "2022-04"; volume: "24"; issue: "4"; pages: "448-460"
COMMENT     Sequence Label: pPB-Ins-U6p-sgRNAentry-EF1Ap-TetOn3G-IRES-Neo
FEATURES             Location/Qualifiers
     source          1..7329
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..67
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        494..742
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     494..514
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     665..684
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        940..1151
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1163..1909
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    join(1948..2522,2523..2525,2526..2534)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2115..2132)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2342..2361
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2535..3329
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2589..2608)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3199..3218
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3336..3384
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   3914..3948
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4439..4455)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4439..4455)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4452..4474)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4463..4479
                     /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(4487..4493,4494..4511,4512..4517))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4532..4553
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4670..4687)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4841..5429)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4921..4940)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5600..6391,6392..6460))
                     /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     6223..6242
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6461..6565)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6591..7046)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6728..6749)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6940..6959
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7173..7195
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7187..7204
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7188..7204
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg tgtaaaattg
       61 acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa
      121 gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt
      181 tatttattta ttaaaaaaaa caaaaactca aaatttcttc tataaagtaa caaaactttt
      241 atgagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg
      301 gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag
      361 ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg
      421 aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaggcctc
      481 cacggccact agtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      541 ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa
      601 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      661 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      721 cttgtggaaa ggacgaaaca ccggagacga tgcgtaggga gatccggaat ctattggccc
      781 gtctccgttt aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt
      841 atcaacttga aaaagtggca ccgagtcggt gcttttttta gcgctctagt taggcgtatc
      901 acctagtaag gatctgcgat cgctccggtg cccgtcagtg ggcagagcgc acatcgccca
      961 cagtccccga gaagttgggg ggaggggtcg gcaattgaac gggtgcctag agaaggtggc
     1021 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     1081 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     1141 ccagaacaca gctcgagcca ccatgtctag actggacaag agcaaagtca taaactctgc
     1201 tctggaatta ctcaatggag tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa
     1261 gctgggagtt gagcagccta ccctgtactg gcacgtgaag aacaagcggg ccctgctcga
     1321 tgccctgcca atcgagatgc tggacaggca tcatacccac tcctgccccc tggaaggcga
     1381 gtcatggcaa gactttctgc ggaacaacgc caagtcatac cgctgtgctc tcctctcaca
     1441 tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca gagaaacagt acgaaaccct
     1501 ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg cactgtacgc
     1561 tctgtccgcc gtgggccact ttacactggg ctgcgtattg gaggaacagg agcatcaagt
     1621 agcaaaagag gaaagagaga cacctaccac cgattctatg cccccacttc tgaaacaagc
     1681 aattgagctg ttcgaccggc agggagccga acctgccttc cttttcggcc tggaactaat
     1741 catatgtggc ctggagaaac agctaaagtg cgaaagcggc gggccgaccg acgcccttga
     1801 cgattttgac ttagacatgc tcccagccga tgcccttgac gactttgacc ttgatatgct
     1861 gcctgctgac gctcttgacg attttgacct tgacatgctc cccgggtaag gccgctcgat
     1921 aagcttgata tcgaattccg cccccccccc ctctccctcc ccccccccta acgttactgg
     1981 ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt
     2041 gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc
     2101 taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc
     2161 agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg
     2221 gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc
     2281 tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa
     2341 atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg
     2401 tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa
     2461 aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata
     2521 atatggccac aaccatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     2581 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     2641 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     2701 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     2761 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     2821 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     2881 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     2941 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3001 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3061 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     3121 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     3181 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     3241 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     3301 tctatcgcct tcttgacgag ttcttctgag gggatcggca ataaaaagac agaataaaac
     3361 gcacgggtgt tgggtcgttt gttcggatcc gggcccattg gtatggcttt ttccccgtat
     3421 ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg
     3481 tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga
     3541 gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg
     3601 taattacatc cctgggggct ttgggggggg gctgtccctg atatctataa caagaaaata
     3661 tatatataat aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag
     3721 ttaaatctta aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta
     3781 tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg
     3841 gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa
     3901 tatttcaaga atgcatgcgt caattttacg cagactatct ttctagggtt aatctagctg
     3961 catcaggatc atatcgtcgg gtcttttttc cggctcagtc atcgcccaag ctggcgctat
     4021 ctgggcatcg gggaggaaga agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa
     4081 gagtttgccg aggatgactg ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg
     4141 atgattcggg aaggtgtggc catgcacgcc tttaacggtg aactgttcgt tcaggccacc
     4201 tgggatacca gttcgtcgcg gcttttccgg acacagttcc ggatggtcag cccgaagcgc
     4261 atcagcaacc cgaacaatac cggcgacagc cggaactgcc gtgccggtgt gcagattaat
     4321 gacagcggtg cggcgctggg atattacgtc agcgaggacg ggtatcctgg ctggatgccg
     4381 cagaaatgga catggatacc ccgtgagtta cccggcgggc gcgcttggcg taatcatggt
     4441 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4501 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4561 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4621 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4681 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4741 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4801 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4861 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4921 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4981 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5041 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5101 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5161 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5221 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5281 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5341 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5401 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5461 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5521 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5581 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5641 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5701 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5761 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5821 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5881 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5941 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6001 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6061 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6121 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6181 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6241 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6301 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6361 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6421 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6481 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6541 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     6601 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     6661 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     6721 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     6781 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     6841 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     6901 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     6961 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     7021 gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga
     7081 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     7141 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     7201 cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg gaggacgggc agactcgcgg
     7261 tgcaaatgtg ttttacagcg tgatggagca gatgaagatg ctcgacacgc tgcagaacac
     7321 gcagctaga
//
