LOCUS       sequence-442528-        7288 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            HOXA5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7288)
  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 7288)
  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-cERP2-HOXA5
FEATURES             Location/Qualifiers
     source          1..7288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    16..34
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    52..70
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    88..106
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    124..142
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    160..178
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    196..214
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    232..250
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     277..301
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    367..391
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1202..1226)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1228..1254
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1421..1555
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1827..1861
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1872..1889)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1872..1888)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1881..1903)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2097..2116)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2218..2240
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2279..2297)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2365..2469
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2470..2538,2539..3330)
                     /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(2688..2707)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3501..4089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3990..4009
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4243..4260
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4466..4487
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4502..4507,4508..4525,4526..4532)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4540..4556
                     /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     4545..4567
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4564..4580
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4564..4580
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4606..4668
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4844..4925
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5642..5698)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(5708..6451)
                     /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(6452..6475)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6475..6484
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6504..6715)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6716..7095
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tcacctcgag tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca
       61 gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt
      121 tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac
      181 gtatctacag tttactccct atcagtgata gagaacgtat atccagttta ctccctatca
      241 gtgatagaga acgtataagc ttgcctatat aagcagagct cgtttagtga accgtcagat
      301 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
      361 cctaccacaa gtttgtacaa aaaagttggc atgagctctt attttgtaaa ctcattttgc
      421 ggtcgctatc caaatggccc ggactaccag ttgcataatt atggagatca tagttccgtg
      481 agcgagcaat tcagggactc ggcgagcatg cactccggca ggtacggcta cggctacaat
      541 ggcatggatc tcagcgtcgg ccgctcgggc tccggccact ttggctccgg agagcgcgcc
      601 cgcagctacg ctgccagcgc cagcgcggcg cccgccgagc ccaggtacag ccagccggcc
      661 acgtccacgc actctcctca gcccgatccg ctgccctgct ccgccgtggc cccctcgccc
      721 ggcagcgaca gccaccacgg cgggaaaaac tccctaagca actccagcgg cgcctcggcc
      781 gacgccggca gcacccacat cagcagcaga gagggggttg gcacggcgtc cggagccgag
      841 gaggacgccc ctgccagcag cgagcaggcg agtgcgcaga gcgagccgag cccggcgccg
      901 cccgcccaac cccagatcta cccctggatg cgcaagctgc acataagtca tgacaacata
      961 ggcggcccgg aaggcaaaag ggcccggacg gcctacacgc gctaccagac cctggagctg
     1021 gagaaggagt tccacttcaa ccgttacctg acccgcagaa ggaggattga aatagcacat
     1081 gctctttgcc tctccgagag acaaattaaa atctggttcc aaaaccggag aatgaagtgg
     1141 aaaaaagata ataagctgaa aagcatgagc atggccgcgg caggaggggc cttccgtccc
     1201 tgcccaactt tcttgtacaa agtggtatac ccatacgatg ttccagatta cgcttagtaa
     1261 tgagaaatac tcagaagatg tcactcacca tagtccgcag tgaaagataa atgatcgatg
     1321 agcgctgcat ttttacgttt tagagctaga aatagcaagt aggccactga gcaagaacta
     1381 tgaatgagat ctttagacat attgcggccg ctttctccgc gatccagaca tgataagata
     1441 cattgatgag tttggacaaa ccaaaactag aatgcagtga aaaaaatgcc ttatttgtga
     1501 aatttgtgat gctattgcct tatttgtaac cattataagc tgcaataaac aagttatcta
     1561 taacaagaaa atatatatat aataagttat cacgtaagta gaacatgaaa taacaatata
     1621 attatcgtat gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt cattttgact
     1681 cacgcggtcg ttatagttca aaatcagtga cacttaccgc attgacaagc acgcctcacg
     1741 ggagctccaa gcggcgactg agatgtccta aatgcacagc gacggattcg cgctatttag
     1801 aaagagagag caatatttca agaatgcatg cgtcaatttt acgcagacta tctttctagg
     1861 gttaagaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     1921 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     1981 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg
     2041 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     2101 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     2161 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac catgtctccg
     2221 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acacgcgagt tcgaaagggc
     2281 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     2341 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2401 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2461 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2521 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2581 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2641 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2701 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2761 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2821 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2881 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2941 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3001 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3061 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3121 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3181 cgtggttcac gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3241 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3301 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3361 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3421 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3481 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3541 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3601 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     3661 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3721 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3781 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3841 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3901 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3961 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4021 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4081 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4141 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4201 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4261 gaagcggatg agcgcccaat acgcaaaccg cctctcaccg cgcgttggcc gattcattaa
     4321 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaaaccgc
     4381 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     4441 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     4501 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     4561 acacaggaaa cagctatgac catgattacg ccaaggtcga cttaacccta gaaagataat
     4621 catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcggt
     4681 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact
     4741 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa
     4801 aacaaaaact caaaatttct tctataaagt aacaaaactt ttagcagtga aaaaaatgct
     4861 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     4921 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     4981 ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat cctctggaga
     5041 ttcatgcgcc aggttttctg gtcatgcacc acgttcgtgg gccctcagga acctccacgt
     5101 cagcggtaac tgtgaagccc agtcgctcgt aaaaaggaag attccgggga gcactggttt
     5161 cgaggaacgc cggcacgcca gcccgttccg cggcctcaac accaggaaga acgacagcgc
     5221 ttcccaaacc tttaccttgg tgatctgggc tgacaccgac agtggcaaga aaccacgcag
     5281 gctcctttgg tctatgagga gccagcaagc cctccatttg ctgttgtgcc gccaacctac
     5341 tccctgacag ctcggccatc cgcggaccta tttccgcgaa cactgcgccg gcttcgacgc
     5401 tttccggagt agtccagacc gcaactgcgg caccatcgtc cgcaacccaa actttaccaa
     5461 tgtccagacc cacgcgcgta agaaacagct cttgcaactc agttaccctc tctatgtgtc
     5521 tgtccggatc tacggtatgg cgggtcgctg gataatctgc aaatgctgcg gcaagtgttc
     5581 ggacagcccg tggtacatcg tcacgggtag cgagcctaac agtgggctta tattccgtca
     5641 tggggccggg gttctcctcc acgtcgccgg cctgcttcag caggctgaag ttggtggcgc
     5701 cgctgccccc ggggagcatg tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat
     5761 caaggtcaaa gtcgtcaagg gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg
     5821 cgtcggtcgg cccgccgctt tcgcacttta gctgtttctc caggccacat atgattagtt
     5881 ccaggccgaa aaggaaggca ggttcggctc cctgccggtc gaacagctca attgcttgtt
     5941 tcagaagtgg gggcatagaa tcggtggtag gtgtctctct ttcctctttt gctacttgat
     6001 gctcctgttc ctccaatacg cagcccagtg taaagtggcc cacggcggac agagcgtaca
     6061 gtgcgttctc cagggagaag ccttgctgac acaggaacgc gagctgattt tccagggttt
     6121 cgtactgttt ctctgttggg cgggtgccga gatgcacttt agccccgtca cgatgtgaga
     6181 ggagagcaca gcggtatgac ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt
     6241 ccagggggca ggagtgggta tgatgcctgt ccagcatctc gattggcagg gcatcgagca
     6301 gggcccgctt gttcttcacg tgccagtaca gggtaggctg ctcaactccc agcttttgag
     6361 cgagtttcct tgtcgtcagg ccttcgatac cgactccatt gagtaattcc agagcagagt
     6421 ttatgacttt gctcttgtcc agtctagaca tcttatcgtc atcgtctttg taatccatgg
     6481 tggcggatcc cgcgtcacga cacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg
     6541 ttcacggcga ctactgcact tatatacggt tctcccccac cctcgggaaa aaggcggagc
     6601 cagtacacga catcactttc ccagtttacc ccgcgccacc ttctctaggc accggttcaa
     6661 ttgccgaccc ctccccccaa cttctcgggc actgtgggcc atgtgcgctc tgccccatgg
     6721 taatagccat gactaatacg tagatgtact gccaagtagg aaagtgccat aaggtcatgt
     6781 actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg ggcgtacttg
     6841 gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac tccacccatt
     6901 gacgtcaatg gaaagtgcct attggcgtta ctatgggaac atacgtcatt attgacgtca
     6961 atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt tatgtaacgc
     7021 ggaactccat atatgggcta tgaactaatg accccgtaat tgattactat taataactag
     7081 tcaataatca atgtcataag ctctaatcgc ataatcgagc taagagcgat taatatgctt
     7141 agctagctta gctaagctag atttctctat tctcccacaa tatatagcag ctattagctt
     7201 agcagtccga atacgatcgt ccacgatcca taagcgtaac acatttgagt agcgactgac
     7261 gggcaccgga gccactcgag tggaatta
//
