LOCUS       sequence-437991-        7561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            HOXB5, with hygromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7561)
  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 7561)
  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-cT3G-cERH-HOXB5
FEATURES             Location/Qualifiers
     source          1..7561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     278..302
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    368..392
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1203..1227)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1229..1255
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1269..1403
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1675..1709
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1720..1737)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1720..1736)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1729..1751)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1945..1964)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2066..2088
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2127..2145)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2213..2317
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2318..2386,2387..3178)
                     /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(2536..2555)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3349..3937
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3838..3857
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4091..4108
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4314..4335
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4350..4355,4356..4373,4374..4380)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4388..4404
                     /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     4393..4415
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4412..4428
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4412..4428
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4454..4516
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4692..4773
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4890..5915)
                     /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"
     CDS             complement(5982..6725)
                     /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"
     CDS             complement(6726..6749)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6749..6758
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6778..6989)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6990..7369
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag cttgcctata taagcagagc tcgtttagtg aaccgtcaga
      301 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      361 gcctaccaca agtttgtaca aaaaagttgg caccatgagc tcgtactttg taaactcctt
      421 ctcggggcgt tatccaaatg gcccggacta tcagttgcta aattatggca gtggcagctc
      481 tctgagcggc tcttacaggg atcccgctgc catgcacacc ggctcttacg gctacaatta
      541 caatgggatg gacctcagcg tcaaccgctc ctcggcctcc tccagccact ttggggcggt
      601 gggcgagagc tcgcgcgcct tccccgcgcc cgcccaggag ccccgcttca ggcaagcggc
      661 ttcgagctgc tccctgtcct cgcccgagtc cctgccctgc accaacggcg acagccacgg
      721 cgccaagccc tctgcttcgt ccccctccga ccaggcgacc tcagccagct ccagcgccaa
      781 tttcaccgaa atagacgagg ccagcgcgtc ctcggagcct gaggaagcgg caagccagct
      841 aagcagcccc agcctagctc gggcgcagcc agagcccatg gccacctcca cagccgcgcc
      901 cgaggggcag actccgcaaa tattcccctg gatgaggaag cttcacatca gccatgatat
      961 gaccgggccg gacgggaaaa gggcccggac cgcgtatacc cgctaccaga ccctggagct
     1021 ggaaaaggag ttccacttca accgctacct gacccggcga cggcgcatcg agatcgccca
     1081 cgcactctgc ctgtccgagc gccagatcaa gatctggttc cagaaccggc gcatgaagtg
     1141 gaagaaggac aacaaattga aaagtatgag cctggctaca gctggcagcg ccttccagcc
     1201 cttgccaact ttcttgtaca aagtggtata cccatacgat gttccagatt acgcttaact
     1261 ttcaccgcga tccagacatg ataagataca ttgatgagtt tggacaaacc aaaactagaa
     1321 tgcagtgaaa aaaatgcctt atttgtgaaa tttgtgatgc tattgcctta tttgtaacca
     1381 ttataagctg caataaacaa gttatctata acaagaaaat atatatataa taagttatca
     1441 cgtaagtaga acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg
     1501 taaaagataa tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca
     1561 cttaccgcat tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa
     1621 tgcacagcga cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg
     1681 tcaattttac gcagactatc tttctagggt taagaattca ctggccgtcg ttttacaacg
     1741 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     1801 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     1861 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     1921 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     1981 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2041 ttacagacaa gctgtgacca tgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     2101 tcaccgaaac acgcgagttc gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2161 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     2221 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2281 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     2341 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2401 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     2461 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     2521 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     2581 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     2641 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     2701 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     2761 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     2821 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     2881 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     2941 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3001 tattgctgat aaatctggag ccggtgagcg tggttcacgc ggtatcattg cagcactggg
     3061 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3121 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3181 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3241 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3301 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3361 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3421 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3481 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3541 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3601 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3661 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3721 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3781 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3841 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3901 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3961 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4021 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4081 gaccgagcgc agcgagtcag tgagcgagga agcggatgag cgcccaatac gcaaaccgcc
     4141 tctcaccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     4201 agcgggcagt gagcgcaacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     4261 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     4321 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     4381 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     4441 aaggtcgact taaccctaga aagataatca tattgtgacg tacgttaaag ataatcatgc
     4501 gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag gtttatttat taatttgaat
     4561 agatattaag ttttattata tttacactta catactaata ataaattcaa caaacaattt
     4621 atttatgttt atttatttat taaaaaaaaa caaaaactca aaatttcttc tataaagtaa
     4681 caaaactttt agcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4741 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4801 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     4861 ggtatggctg attatgatcc tctggagatc tattcctttg ccctcggacg agtgctgggg
     4921 cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac ggccgcgctt
     4981 ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat tgcgtcgcat
     5041 cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg atagagttgg
     5101 tcaagaccaa tgcggagcat atacgcccgg agccgcggcg atcctgcaag ctccggatgc
     5161 ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct ccagaagaag
     5221 atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc aatgaccgct
     5281 gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg cacgaggtgc
     5341 cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg cgcgacggac
     5401 gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc agcaatcgcg
     5461 catatgaaat cacgccacgt agtgtattga ccgattcctt gcggtccgaa tgggccgaac
     5521 ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca tggcctccgc gaccggctgc
     5581 agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg tgcacggcgg
     5641 gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc cggaatcggg
     5701 agcgcggccg atgcaaagtg ccgatgaaca taacgatctt tgtagaaacc atcggcgcag
     5761 ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc acgagattct
     5821 tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat cagaaacttc
     5881 tcgacagacg tcgcggtgag ttcaggcttt ttcatggggc cggggttctc ctccacttcg
     5941 ccggcctgct tcagcaggct gaagttggtg gcgccgctgc ccccggggag catgtcaagg
     6001 tcaaaatcgt caagagcgtc agcaggcagc atatcaaggt caaagtcgtc aagggcatcg
     6061 gctgggagca tgtctaagtc aaaatcgtca agggcgtcgg tcggcccgcc gctttcgcac
     6121 tttagctgtt tctccaggcc acatatgatt agttccaggc cgaaaaggaa ggcaggttcg
     6181 gctccctgcc ggtcgaacag ctcaattgct tgtttcagaa gtgggggcat agaatcggtg
     6241 gtaggtgtct ctctttcctc ttttgctact tgatgctcct gttcctccaa tacgcagccc
     6301 agtgtaaagt ggcccacggc ggacagagcg tacagtgcgt tctccaggga gaagccttgc
     6361 tgacacagga acgcgagctg attttccagg gtttcgtact gtttctctgt tgggcgggtg
     6421 ccgagatgca ctttagcccc gtcacgatgt gagaggagag cacagcggta tgacttggcg
     6481 ttgttccgca gaaagtcttg ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc
     6541 ctgtccagca tctcgattgg cagggcatcg agcagggccc gcttgttctt cacgtgccag
     6601 tacagggtag gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt caggccttcg
     6661 ataccgactc cattgagtaa ttccagagca gagtttatga ctttgctctt gtccagtcta
     6721 gacatcttat cgtcatcgtc tttgtaatcc atggtggcgg atcccgcgtc acgacacctg
     6781 tgttctggcg gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg cacttatata
     6841 cggttctccc ccaccctcgg gaaaaaggcg gagccagtac acgacatcac tttcccagtt
     6901 taccccgcgc caccttctct aggcaccggt tcaattgccg acccctcccc ccaacttctc
     6961 gggcactgtg ggccatgtgc gctctgcccc atggtaatag ccatgactaa tacgtagatg
     7021 tactgccaag taggaaagtg ccataaggtc atgtactggg cataatgcca ggcgggccat
     7081 ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc
     7141 aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt gcctattggc
     7201 gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag
     7261 ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact
     7321 aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca taagctctaa
     7381 tcgcataatc gagctaagag cgattaatat gcttagctag cttagctaag ctagatttct
     7441 ctattctccc acaatatata gcagctatta gcttagcagt ccgaatacga tcgtccacga
     7501 tccataagcg taacacattt gagtagcgac tgacgggcac cggagccact cgagtggaat
     7561 t
//
