LOCUS       sequence-377556-        7888 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            KLF4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7888)
  AUTHORS   Pierson Smela MD, Kramme CC, Fortuna PRJ, Adams JL, Su R, Dong E, 
            Kobayashi M, Brixi G, Kavirayuni VS, Tysinger E, Kohman RE, Shioda 
            T, Chatterjee P, Church GM
  TITLE     Directed differentiation of human iPSCs to functional ovarian 
            granulosa-like cells via transcription factor overexpression.
  JOURNAL   Elife. 2023 Feb 21;12:e83291. doi: 10.7554/eLife.83291.
  PUBMED    36803359
REFERENCE   2  (bases 1 to 7888)
  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: "Elife."; 
            date: "2023-02-21"; volume: "12:e83291. doi"; pages: " 
            10.7554/eLife.83291"
COMMENT     Sequence Label: pPB-cT3G-cERP2-KLF4
FEATURES             Location/Qualifiers
     source          1..7888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..45
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    111..135
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1546..1570)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1572..1598
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1765..1899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2171..2205
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2216..2233)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2216..2232)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2225..2247)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2441..2460)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2562..2584
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2623..2641)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2709..2813
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2814..2882,2883..3674)
                     /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(3032..3051)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3845..4433
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4334..4353
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4587..4604
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4810..4831
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4846..4851,4852..4869,4870..4876)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4884..4900
                     /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     4889..4911
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4908..4924
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4908..4924
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4950..5012
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5188..5269
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5986..6042)
                     /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(6052..6795)
                     /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(6796..6819)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6819..6828
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6848..7059)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        7060..7439
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7648..7666
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7684..7702
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7720..7738
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7756..7774
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7792..7810
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7828..7846
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7864..7882
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 aagcttgcct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat
       61 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctacc acaagtttgt
      121 acaaaaaagt tggcatggct gtcagcgacg cgctgctccc atctttctcc acgttcgcgt
      181 ctggcccggc gggaagggag aagacactgc gtcaagcagg tgccccgaat aaccgctggc
      241 gggaggagct ctcccacatg aagcgacttc ccccagtgct tcccggccgc ccctatgacc
      301 tggcggcggc gaccgtggcc acagacctgg agagcggcgg agccggtgcg gcttgcggcg
      361 gtagcaacct ggcgccccta cctcggagag agaccgagga gttcaacgat ctcctggacc
      421 tggactttat tctctccaat tcgctgaccc atcctccgga gtcagtggcc gccaccgtgt
      481 cctcgtcagc gtcagcctcc tcttcgtcgt cgccgtcgag cagcggccct gccagcgcgc
      541 cctccacctg cagcttcacc tatccgatcc gggccgggaa cgacccgggc gtggcgccgg
      601 gcggcacggg cggaggcctc ctctatggca gggagtccgc tccccctccg acggctccct
      661 tcaacctggc ggacatcaac gacgtgagcc cctcgggcgg cttcgtggcc gagctcctgc
      721 ggccagaatt ggacccggtg tacattccgc cgcagcagcc gcagccgcca ggtggcgggc
      781 tgatgggcaa gttcgtgctg aaggcgtcgc tgagcgcccc tggcagcgag tacggcagcc
      841 cgtcggtcat cagcgtcagc aaaggcagcc ctgacggcag ccacccggtg gtggtggcgc
      901 cctacaacgg cgggccgccg cgcacgtgcc ccaagatcaa gcaggaggcg gtctcttcgt
      961 gcacccactt gggcgctgga ccccctctca gcaatggcca ccggccggct gcacacgact
     1021 tccccctggg gcggcagctc cccagcagga ctaccccgac cctgggtctt gaggaagtgc
     1081 tgagcagcag ggactgtcac cctgccctgc cgcttcctcc cggcttccat ccccacccgg
     1141 ggcccaatta cccatccttc ctgcccgatc agatgcagcc gcaagtcccg ccgctccatt
     1201 accaagagct catgccaccc ggttcctgca tgccagagga gcccaagcca aagaggggaa
     1261 gacgatcgtg gccccggaaa aggaccgcca cccacacttg tgattacgcg ggctgcggca
     1321 aaacctacac aaagagttcc catctcaagg cacacctgcg aacccacaca ggtgagaaac
     1381 cttaccactg tgactgggac ggctgtggat ggaaattcgc ccgctcagat gaactgacca
     1441 ggcactaccg taaacacacg gggcaccgcc cgttccagtg ccaaaaatgc gaccgagcat
     1501 tttccaggtc ggaccacctc gccttacaca tgaagaggca tttttaccca actttcttgt
     1561 acaaagtggt atacccatac gatgttccag attacgctta gtaatgagaa atactcagaa
     1621 gatgtcactc accatagtcc gcagtgaaag ataaatgatc caactgggaa atcaacaagt
     1681 gttttagagc tagaaatagc aagtaggcca ctgagcaaga actatgaatg agatctttag
     1741 acatattgcg gccgctttct ccgcgatcca gacatgataa gatacattga tgagtttgga
     1801 caaaccaaaa ctagaatgca gtgaaaaaaa tgccttattt gtgaaatttg tgatgctatt
     1861 gccttatttg taaccattat aagctgcaat aaacaagtta tctataacaa gaaaatatat
     1921 atataataag ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta
     1981 aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag
     2041 ttcaaaatca gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg
     2101 actgagatgt cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat
     2161 ttcaagaatg catgcgtcaa ttttacgcag actatctttc tagggttaag aattcactgg
     2221 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     2281 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     2341 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
     2401 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
     2461 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2521 tgctcccggc atccgcttac agacaagctg tgaccatgtc tccgggagct gcatgtgtca
     2581 gaggttttca ccgtcatcac cgaaacacgc gagttcgaaa gggcctcgtg atacgcctat
     2641 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     2701 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2761 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     2821 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2881 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2941 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     3001 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     3061 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     3121 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     3181 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     3241 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3301 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     3361 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3421 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3481 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggt tcacgcggta
     3541 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3601 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3661 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     3721 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3781 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3841 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3901 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3961 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     4021 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4081 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4141 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4201 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4261 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4321 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4381 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4441 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4501 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4561 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gatgagcgcc
     4621 caatacgcaa accgcctctc accgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     4681 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaaa ccgcctctcc ccgcgcgttg
     4741 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     4801 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     4861 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     4921 tgaccatgat tacgccaagg tcgacttaac cctagaaaga taatcatatt gtgacgtacg
     4981 ttaaagataa tcatgcgtaa aattgacgca tgtgttttat cggtctgtat atcgaggttt
     5041 atttattaat ttgaatagat attaagtttt attatattta cacttacata ctaataataa
     5101 attcaacaaa caatttattt atgtttattt atttattaaa aaaaaacaaa aactcaaaat
     5161 ttcttctata aagtaacaaa acttttagca gtgaaaaaaa tgctttattt gtgaaatttg
     5221 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     5281 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     5341 aaacctctac aaatgtggta tggctgatta tgatcctctg gagattcatg cgccaggttt
     5401 tctggtcatg caccacgttc gtgggccctc aggaacctcc acgtcagcgg taactgtgaa
     5461 gcccagtcgc tcgtaaaaag gaagattccg gggagcactg gtttcgagga acgccggcac
     5521 gccagcccgt tccgcggcct caacaccagg aagaacgaca gcgcttccca aacctttacc
     5581 ttggtgatct gggctgacac cgacagtggc aagaaaccac gcaggctcct ttggtctatg
     5641 aggagccagc aagccctcca tttgctgttg tgccgccaac ctactccctg acagctcggc
     5701 catccgcgga cctatttccg cgaacactgc gccggcttcg acgctttccg gagtagtcca
     5761 gaccgcaact gcggcaccat cgtccgcaac ccaaacttta ccaatgtcca gacccacgcg
     5821 cgtaagaaac agctcttgca actcagttac cctctctatg tgtctgtccg gatctacggt
     5881 atggcgggtc gctggataat ctgcaaatgc tgcggcaagt gttcggacag cccgtggtac
     5941 atcgtcacgg gtagcgagcc taacagtggg cttatattcc gtcatggggc cggggttctc
     6001 ctccacgtcg ccggcctgct tcagcaggct gaagttggtg gcgccgctgc ccccggggag
     6061 catgtcaagg tcaaaatcgt caagagcgtc agcaggcagc atatcaaggt caaagtcgtc
     6121 aagggcatcg gctgggagca tgtctaagtc aaaatcgtca agggcgtcgg tcggcccgcc
     6181 gctttcgcac tttagctgtt tctccaggcc acatatgatt agttccaggc cgaaaaggaa
     6241 ggcaggttcg gctccctgcc ggtcgaacag ctcaattgct tgtttcagaa gtgggggcat
     6301 agaatcggtg gtaggtgtct ctctttcctc ttttgctact tgatgctcct gttcctccaa
     6361 tacgcagccc agtgtaaagt ggcccacggc ggacagagcg tacagtgcgt tctccaggga
     6421 gaagccttgc tgacacagga acgcgagctg attttccagg gtttcgtact gtttctctgt
     6481 tgggcgggtg ccgagatgca ctttagcccc gtcacgatgt gagaggagag cacagcggta
     6541 tgacttggcg ttgttccgca gaaagtcttg ccatgactcg ccttccaggg ggcaggagtg
     6601 ggtatgatgc ctgtccagca tctcgattgg cagggcatcg agcagggccc gcttgttctt
     6661 cacgtgccag tacagggtag gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt
     6721 caggccttcg ataccgactc cattgagtaa ttccagagca gagtttatga ctttgctctt
     6781 gtccagtcta gacatcttat cgtcatcgtc tttgtaatcc atggtggcgg atcccgcgtc
     6841 acgacacctg tgttctggcg gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg
     6901 cacttatata cggttctccc ccaccctcgg gaaaaaggcg gagccagtac acgacatcac
     6961 tttcccagtt taccccgcgc caccttctct aggcaccggt tcaattgccg acccctcccc
     7021 ccaacttctc gggcactgtg ggccatgtgc gctctgcccc atggtaatag ccatgactaa
     7081 tacgtagatg tactgccaag taggaaagtg ccataaggtc atgtactggg cataatgcca
     7141 ggcgggccat ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg
     7201 atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt
     7261 gcctattggc gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt
     7321 gggcggtcag ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg
     7381 gctatgaact aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca
     7441 taagctctaa tcgcataatc gagctaagag cgattaatat gcttagctag cttagctaag
     7501 ctagatttct ctattctccc acaatatata gcagctatta gcttagcagt ccgaatacga
     7561 tcgtccacga tccataagcg taacacattt gagtagcgac tgacgggcac cggagccact
     7621 cgagtggaat tatcacctcg agtttactcc ctatcagtga tagagaacgt atgaagagtt
     7681 tactccctat cagtgataga gaacgtatgc agactttact ccctatcagt gatagagaac
     7741 gtataaggag tttactccct atcagtgata gagaacgtat gaccagttta ctccctatca
     7801 gtgatagaga acgtatctac agtttactcc ctatcagtga tagagaacgt atatccagtt
     7861 tactccctat cagtgataga gaacgtat
//
