LOCUS       sequence-442404-        7936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBac transposon plasmid for doxycycline inducible expression of 
            ANHX.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7936)
  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 7936)
  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-ANHX
FEATURES             Location/Qualifiers
     source          1..7936
                     /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(1851..1875)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1877..1903
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2070..2204
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2476..2510
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2521..2538)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2521..2537)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2530..2552)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2746..2765)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2867..2889
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2928..2946)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3014..3118
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3119..3187,3188..3979)
                     /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(3337..3356)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4150..4738
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4639..4658
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4892..4909
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5115..5136
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5151..5156,5157..5174,5175..5181)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5189..5205
                     /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     5194..5216
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5213..5229
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5213..5229
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5255..5317
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5493..5574
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6291..6347)
                     /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(6357..7100)
                     /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(7101..7124)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7124..7133
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7153..7364)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7365..7744
                     /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 aaaaagcagg ctgccaccat gcagagcttc ctgactctgc
      421 tgaaggagca tgaggacacc tgtgcacccc cggcggagct ggtgaccctt gcgggcagac
      481 tgtgccggga cttccaggat gaccttgccc aactgcagcc tttggtcaca gccattctgg
      541 acagccagct ccgcctgcat ctcctggaca acgcagatgt ggccctggcg tgcgcccgtg
      601 tcctggacca gcaggagcag cagcaggcgg cttgccgcct cctggagggg tgccaggtgc
      661 cgggaggcag ccaggagtta gtgcagctct ggaacgacat ccactaccgt ctggtcatga
      721 ggaggctggg cgtggctgcg ctcaccccgg tgcagaagtt ccgctgcagg aagaggaacc
      781 ccccgccccc ctccctctgc ccagaggggc tgaagagccg gaacttcccc agagaggttc
      841 gtgagaagct gcacaatttc gctgtggggg tgaacaccaa ccccagcaag gctgagaggg
      901 agaacttggc attggagacg agcttgaccc ctgagcaggt gtacaactgg tttgccaatt
      961 accggcgccg ccaaagagcc cttccccagc acatgaagcc agcccagcag gccacagctg
     1021 aagaccctgg tgcgagggag aggggtcctg acctcctgca gccctcaggc aacccccgtg
     1081 ttgactctgg gtttgtggac aggcctcagt ggtcagagga acgtgaggaa aaggggcctc
     1141 cacagtcccc acagaccacc caaggaccat gggagccact ggccttagcc ccggactttc
     1201 ccgcagatga gacagtctca aagccactgg atgtcaggtc tctgccaggt ggcgagatgt
     1261 accaggaggg gcctggccat gatcctgcca ccctccccct cttctgccct ggccctggcc
     1321 tctgccctct ggctgctggc aatgacatgc tgaacccctc tctggctgcc cctgaatctt
     1381 ggctgatgtc tcttgcactg gcgtcctcca aggaagtttc cttccagact gggcagctgg
     1441 tccacagcca tgggctggac ttcatgatgg gccctgcaga caccgctgtg gctgtgtcca
     1501 ttgccaccct tggagatccc agccctacag ggttttctgg cccccccagc ggccatcccc
     1561 agagcgtgca attggaggag ggtctgggca caagcagtgg acggacagag ctacgggtgg
     1621 gcagcttcct ggtgacacag cccccactgc aagctcctga attcatcctc acccagagcc
     1681 ctccagagct ggccccagcc ccatctgcct tccccggccc tgtgtctgcc atggagctga
     1741 gccaggccct gccctccagc caggtgcagt gttctgatag ccaggcctct ggtgatgcct
     1801 tctggggagc caggatgctc cttgagtttt cagggagcag cctgggctga acccagcttt
     1861 cttgtacaaa gtggtatacc catacgatgt tccagattac gcttagtaat gagaaatact
     1921 cagaagatgt cactcaccat agtccgcagt gaaagataaa tgatcttatg actttgactt
     1981 gacaagtttt agagctagaa atagcaagta ggccactgag caagaactat gaatgagatc
     2041 tttagacata ttgcggccgc tttctccgcg atccagacat gataagatac attgatgagt
     2101 ttggacaaac caaaactaga atgcagtgaa aaaaatgcct tatttgtgaa atttgtgatg
     2161 ctattgcctt atttgtaacc attataagct gcaataaaca agttatctat aacaagaaaa
     2221 tatatatata ataagttatc acgtaagtag aacatgaaat aacaatataa ttatcgtatg
     2281 agttaaatct taaaagtcac gtaaaagata atcatgcgtc attttgactc acgcggtcgt
     2341 tatagttcaa aatcagtgac acttaccgca ttgacaagca cgcctcacgg gagctccaag
     2401 cggcgactga gatgtcctaa atgcacagcg acggattcgc gctatttaga aagagagagc
     2461 aatatttcaa gaatgcatgc gtcaatttta cgcagactat ctttctaggg ttaagaattc
     2521 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     2581 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     2641 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
     2701 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga
     2761 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     2821 ttgtctgctc ccggcatccg cttacagaca agctgtgacc atgtctccgg gagctgcatg
     2881 tgtcagaggt tttcaccgtc atcaccgaaa cacgcgagtt cgaaagggcc tcgtgatacg
     2941 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     3001 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3061 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     3121 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3181 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     3241 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     3301 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     3361 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     3421 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     3481 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     3541 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     3601 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3661 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3721 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3781 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggttcacg
     3841 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3901 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3961 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4021 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4081 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4141 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4201 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4261 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     4321 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4381 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4441 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4501 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4561 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4621 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4681 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4741 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4801 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4861 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggatga
     4921 gcgcccaata cgcaaaccgc ctctcaccgc gcgttggccg attcattaat gcagctggca
     4981 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaaaccgcc tctccccgcg
     5041 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     5101 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     5161 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     5221 agctatgacc atgattacgc caaggtcgac ttaaccctag aaagataatc atattgtgac
     5281 gtacgttaaa gataatcatg cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga
     5341 ggtttattta ttaatttgaa tagatattaa gttttattat atttacactt acatactaat
     5401 aataaattca acaaacaatt tatttatgtt tatttattta ttaaaaaaaa acaaaaactc
     5461 aaaatttctt ctataaagta acaaaacttt tagcagtgaa aaaaatgctt tatttgtgaa
     5521 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     5581 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     5641 aagtaaaacc tctacaaatg tggtatggct gattatgatc ctctggagat tcatgcgcca
     5701 ggttttctgg tcatgcacca cgttcgtggg ccctcaggaa cctccacgtc agcggtaact
     5761 gtgaagccca gtcgctcgta aaaaggaaga ttccggggag cactggtttc gaggaacgcc
     5821 ggcacgccag cccgttccgc ggcctcaaca ccaggaagaa cgacagcgct tcccaaacct
     5881 ttaccttggt gatctgggct gacaccgaca gtggcaagaa accacgcagg ctcctttggt
     5941 ctatgaggag ccagcaagcc ctccatttgc tgttgtgccg ccaacctact ccctgacagc
     6001 tcggccatcc gcggacctat ttccgcgaac actgcgccgg cttcgacgct ttccggagta
     6061 gtccagaccg caactgcggc accatcgtcc gcaacccaaa ctttaccaat gtccagaccc
     6121 acgcgcgtaa gaaacagctc ttgcaactca gttaccctct ctatgtgtct gtccggatct
     6181 acggtatggc gggtcgctgg ataatctgca aatgctgcgg caagtgttcg gacagcccgt
     6241 ggtacatcgt cacgggtagc gagcctaaca gtgggcttat attccgtcat ggggccgggg
     6301 ttctcctcca cgtcgccggc ctgcttcagc aggctgaagt tggtggcgcc gctgcccccg
     6361 gggagcatgt caaggtcaaa atcgtcaaga gcgtcagcag gcagcatatc aaggtcaaag
     6421 tcgtcaaggg catcggctgg gagcatgtct aagtcaaaat cgtcaagggc gtcggtcggc
     6481 ccgccgcttt cgcactttag ctgtttctcc aggccacata tgattagttc caggccgaaa
     6541 aggaaggcag gttcggctcc ctgccggtcg aacagctcaa ttgcttgttt cagaagtggg
     6601 ggcatagaat cggtggtagg tgtctctctt tcctcttttg ctacttgatg ctcctgttcc
     6661 tccaatacgc agcccagtgt aaagtggccc acggcggaca gagcgtacag tgcgttctcc
     6721 agggagaagc cttgctgaca caggaacgcg agctgatttt ccagggtttc gtactgtttc
     6781 tctgttgggc gggtgccgag atgcacttta gccccgtcac gatgtgagag gagagcacag
     6841 cggtatgact tggcgttgtt ccgcagaaag tcttgccatg actcgccttc cagggggcag
     6901 gagtgggtat gatgcctgtc cagcatctcg attggcaggg catcgagcag ggcccgcttg
     6961 ttcttcacgt gccagtacag ggtaggctgc tcaactccca gcttttgagc gagtttcctt
     7021 gtcgtcaggc cttcgatacc gactccattg agtaattcca gagcagagtt tatgactttg
     7081 ctcttgtcca gtctagacat cttatcgtca tcgtctttgt aatccatggt ggcggatccc
     7141 gcgtcacgac acctgtgttc tggcggcaaa cccgttgcga aaaagaacgt tcacggcgac
     7201 tactgcactt atatacggtt ctcccccacc ctcgggaaaa aggcggagcc agtacacgac
     7261 atcactttcc cagtttaccc cgcgccacct tctctaggca ccggttcaat tgccgacccc
     7321 tccccccaac ttctcgggca ctgtgggcca tgtgcgctct gccccatggt aatagccatg
     7381 actaatacgt agatgtactg ccaagtagga aagtgccata aggtcatgta ctgggcataa
     7441 tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac
     7501 acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg
     7561 aaagtgccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg
     7621 tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata
     7681 tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt caataatcaa
     7741 tgtcataagc tctaatcgca taatcgagct aagagcgatt aatatgctta gctagcttag
     7801 ctaagctaga tttctctatt ctcccacaat atatagcagc tattagctta gcagtccgaa
     7861 tacgatcgtc cacgatccat aagcgtaaca catttgagta gcgactgacg ggcaccggag
     7921 ccactcgagt ggaatt
//
