LOCUS       sequence-437872-        7968 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for Shield1-inducible expression of 
            BCL2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7968)
  AUTHORS   Pierson Smela M, Adams J, Ma C, Breimann L, Widocki U, Shioda T, 
            Church GM
  TITLE     Induction of Meiosis from Human Pluripotent Stem Cells
  JOURNAL   bioRxiv 2024.05.31.596483
REFERENCE   2  (bases 1 to 7968)
  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: "bioRxiv 
            2024.05.31.596483"
COMMENT     Sequence Label: pPB-EF1a-HOXB5-DD-hygro
FEATURES             Location/Qualifiers
     source          1..7968
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1204..1222
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1260..1282)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1382..1401
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1595..1617
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1609..1626
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1610..1626
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   1649..1711
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1983..2014
                     /label=rox
                     /bound_moiety="Dre recombinase"
     polyA_signal    2062..2286
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2275..2292
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(2314..3339)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVHRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(3359..3858)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3407..3426)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     3800..3823
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    3908..3939
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        3961..5143
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4192..5134
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5091..5111
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     5145..5169
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    5215..5239
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(6050..6074)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6076..6399
                     /codon_start=1
                     /product="destabilization domain that can be stabilized by 
                     Shield1 in the ProteoTuner(TM) system"
                     /label=DD-C
                     /note="FKBP12 mutant optimized for use as a C-terminal tag"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLGDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQGAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     ELE"
     polyA_signal    6512..6646
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     6590..6609
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   6918..6952
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6978..6994)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6978..6994)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6991..7013)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7002..7018
                     /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(7026..7032,7033..7050,7051..7056))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7071..7092
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7209..7226)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7380..7968)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7460..7479)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gtgacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     1201 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     1261 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     1321 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     1381 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga
     1441 tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg
     1501 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     1561 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     1621 gccagtgaat tcaagcttta ttatttaacc ctagaaagat aatcatattg tgacgtacgt
     1681 taaagataat catgcgtaaa attgacgcat gtgttttatc ggtctgtata tcgaggttta
     1741 tttattaatt tgaatagata ttaagtttta ttatatttac acttacatac taataataaa
     1801 ttcaacaaac aatttattta tgtttattta tttattaaaa aaaaacaaaa actcaaaatt
     1861 tcttctataa agtaacaaaa cttttaaaca ttctctcttt tacaaaaata aacttatttt
     1921 gtactttaaa aacagtcatg ttgtaatcga taataagtaa ttagcctaga tatcggcgcg
     1981 cctaacttta aataattggc attatttaaa gttaagtact ctagtatgtc gagccccagc
     2041 tggttctttc cgcctcagaa gccatagagc ccaccgcatc cccagcatgc ctgctattgt
     2101 cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc
     2161 tactcagaca atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac
     2221 cttccagggt caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag
     2281 gcacagtcga ggctgatcag cgagctctag agcctattcc tttgccctcg gacgagtgct
     2341 ggggcgtcgg tttccactat cggcgagtac ttctacacag ccatcggtcc agacggccgc
     2401 gcttctgcgg gcgatttgtg tacgcccgac agtcccggct ccggatcgga cgattgcgtc
     2461 gcatcgaccc tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc tctgatagag
     2521 ttggtcaaga ccaatgcgga gcatatacgc ccggagccgc ggcgatcctg caagctccgg
     2581 atgcctccgc tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg gcctccagaa
     2641 gaagatgttg gcgacctcgt attgggaatc cccgaacatc gcctcgctcc agtcaatgac
     2701 cgctgttatg cggccattgt ccgtcaggac attgttggag ccgaaatccg cgtgcacgag
     2761 gtgccggact tcggggcagt cctcggccca aagcatcagc tcatcgagag cctgcgcgac
     2821 ggacgcactg acggtgtcgt ccatcacagt ttgccagtga tacacatggg gatcagcaat
     2881 cgcgcatatg aaatcacgcc acgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc
     2941 gaacccgctc gtctggctaa gatcggccgc agcgatcgca tccatggcct ccgcgaccgg
     3001 ctgcagaaca gcgggcagtt cggtttcagg caggtcttgc aacgtgacac cctgtgcacg
     3061 gcgggagatg caataggtca ggctctcgct gaattcccca atgtcaagca cttccggaat
     3121 cgggagcgcg gccgatgcaa agtgccgatg aacataacga tctttgtaga aaccatcggc
     3181 gcagctattt acccgcagga catatccacg ccctcctaca tcgaagctga aagcacgaga
     3241 ttcttcgccc tccgagagct gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa
     3301 cttctcgaca gacgtcgcgg tgagttcagg ctttttcatg gtggcgttgg ctgcaggtcg
     3361 aaaggcccgg agatgaggaa gaggagaaca gcgcggcaga cgtgcgcttt tgaagcgtgc
     3421 agaatgccgg gcctccggag gaccttcggg cgcccgcccc gcccctgagc ccgcccctga
     3481 gcccgccccc ggacccaccc cttcccagcc tctgagccca gaaagcgaag gagcaaagct
     3541 gctattggcc gctgccccaa aggcctaccc gcttccattg ctcagcggtg ctgtccatct
     3601 gcacgagact agtgagacgt gctacttcca tttgtcacgt cctgcacgac gcgagctgcg
     3661 gggcgggggg gaacttcctg actaggggag gagtagaagg tggcgcgaag gggccaccaa
     3721 agaacggagc cggttggcgc ctaccggtgg atgtggaatg tgtgcgaggc cagaggccac
     3781 ttgtgtagcg ccaagtgccc agcggggctg ctaaagcgca tgctccagac tgccttggga
     3841 aaagcgcctc ccctacccgg tagaattaat tcgatatcaa gctgaagttc ctatactttc
     3901 tcatatgtaa ctttaaataa ttggcattat ttaaagttat tacttgtaca gctcgtccat
     3961 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
     4021 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
     4081 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
     4141 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
     4201 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
     4261 acttccacgc ccctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg
     4321 gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc
     4381 ctggcttggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc
     4441 tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt
     4501 ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg
     4561 ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct
     4621 gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt
     4681 gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc
     4741 accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg
     4801 gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt
     4861 tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct
     4921 cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc
     4981 gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat
     5041 gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca
     5101 gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgagagctcg tttagtgaac
     5161 cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acacacaagt
     5221 ttgtacaaaa aagttggcac catgagctcg tactttgtaa actccttctc ggggcgttat
     5281 ccaaatggcc cggactatca gttgctaaat tatggcagtg gcagctctct gagcggctct
     5341 tacagggatc ccgctgccat gcacaccggc tcttacggct acaattacaa tgggatggac
     5401 ctcagcgtca accgctcctc ggcctcctcc agccactttg gggcggtggg cgagagctcg
     5461 cgcgccttcc ccgcgcccgc ccaggagccc cgcttcaggc aagcggcttc gagctgctcc
     5521 ctgtcctcgc ccgagtccct gccctgcacc aacggcgaca gccacggcgc caagccctct
     5581 gcttcgtccc cctccgacca ggcgacctca gccagctcca gcgccaattt caccgaaata
     5641 gacgaggcca gcgcgtcctc ggagcctgag gaagcggcaa gccagctaag cagccccagc
     5701 ctagctcggg cgcagccaga gcccatggcc acctccacag ccgcgcccga ggggcagact
     5761 ccgcaaatat tcccctggat gaggaagctt cacatcagcc atgatatgac cgggccggac
     5821 gggaaaaggg cccggaccgc gtatacccgc taccagaccc tggagctgga aaaggagttc
     5881 cacttcaacc gctacctgac ccggcgacgg cgcatcgaga tcgcccacgc actctgcctg
     5941 tccgagcgcc agatcaagat ctggttccag aaccggcgca tgaagtggaa gaaggacaac
     6001 aaattgaaaa gtatgagcct ggctacagct ggcagcgcct tccagccctt gccaactttc
     6061 ttgtacaaag tggtaggagt gcaggtggaa accatctccc caggagatgg gcgcaccttc
     6121 cccaagcgcg gccagacctg cgtggtgcac tacaccggga tgcttggaga tggaaagaaa
     6181 gttgactcct cccgggacag aaacaagccc tttaagttta tgctaggcaa gcaggaggtg
     6241 atccgaggct gggaagaagg ggttgcccag atgagtgtgg gtcagggagc caaactgact
     6301 atatctccag attatgccta tggtgccact gggcacccag gcatcatccc accacatgcc
     6361 actctcgtgt tcgatgtgga gcttctagaa ctggaatgag aaatactcag aagatgtcac
     6421 tcaccataga gagaccatta atgggtctcc taggccactg agcaagaact atgaatgaga
     6481 tctttagaca tattgcggcc gctttctccg cgatccagac atgataagat acattgatga
     6541 gtttggacaa accaaaacta gaatgcagtg aaaaaaatgc cttatttgtg aaatttgtga
     6601 tgctattgcc ttatttgtaa ccattataag ctgcaataaa caagttatct ataacaagaa
     6661 aatatatata taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta
     6721 tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc
     6781 gttatagttc aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca
     6841 agcggcgact gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga
     6901 gcaatatttc aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaaaaaa
     6961 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     7021 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     7081 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     7141 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgcact
     7201 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     7261 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     7321 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     7381 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     7441 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     7501 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     7561 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     7621 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     7681 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     7741 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     7801 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     7861 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     7921 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaa
//
