LOCUS       sequence-377020-        7531 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            ATF4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7531)
  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 7531)
  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-ATF4
FEATURES             Location/Qualifiers
     source          1..7531
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..42
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    108..132
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             935..952
                     /codon_start=1
                     /product="N-myristoylation signal from Src kinase (Pellman 
                     et al., 1985; Kaplan et al., 1988)"
                     /label=myr
                     /translation="GSSKSK"
     protein_bind    complement(1186..1210)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1212..1238
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1405..1539
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1811..1845
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1856..1873)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1856..1872)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1865..1887)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2081..2100)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2202..2224
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2263..2281)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2349..2453
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2454..2522,2523..3314)
                     /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(2672..2691)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3485..4073
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3974..3993
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4227..4244
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4450..4471
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4486..4491,4492..4509,4510..4516)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4524..4540
                     /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     4529..4551
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4548..4564
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4548..4564
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4590..4652
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4828..4909
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5626..5682)
                     /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(5692..6435)
                     /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(6436..6459)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6459..6468
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6488..6699)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6700..7079
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7288..7306
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7324..7342
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7360..7378
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7396..7414
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7432..7450
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7468..7486
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7504..7522
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 cttgcctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca
       61 cgctgttttg acctccatag aagacaccgg gaccgatcca gcctaccaca agtttgtaca
      121 aaaaagttgg catgaccgaa atgagcttcc tgagcagcga ggtgttggtg ggggacttga
      181 tgtccccctt cgaccagtcg ggtttggggg ctgaagaaag cctaggtctc ttagatgatt
      241 acctggaggt ggccaagcac ttcaaacctc atgggttctc cagcgacaag gctaaggcgg
      301 gctcctccga atggctggct gtggatgggt tggtcagtcc ctccaacaac agcaaggagg
      361 atgccttctc cgggacagat tggatgttgg agaaaatgga tttgaaggag ttcgacttgg
      421 atgccctgtt gggtatagat gacctggaaa ccatgccaga tgaccttctg accacgttgg
      481 atgacacttg tgatctcttt gcccccctag tccaggagac taataagcag cccccccaga
      541 cggtgaaccc aattggccat ctcccagaaa gtttaacaaa acccgaccag gttgccccct
      601 tcaccttctt acaacctctt cccctttccc caggggtcct gtcctccact ccagatcatt
      661 cctttagttt agagctgggc agtgaagtgg atatcactga aggagatagg aagccagact
      721 acactgctta cgttgccatg atccctcagt gcataaagga ggaagacacc ccttcagata
      781 atgatagtgg catctgtatg agcccagagt cctatctggg gtctcctcag cacagcccct
      841 ctaccagggg ctctccaaat aggagcctcc catctccagg tgttctctgt gggtctgccc
      901 gtcccaaacc ttacgatcct cctggagaga agatggtagc agcaaaagta aagggtgaga
      961 aactggataa gaagctgaaa aaaatggagc aaaacaagac agcagccact aggtaccgcc
     1021 agaagaagag ggcggagcag gaggctctta ctggtgagtg caaagagctg gaaaagaaga
     1081 acgaggctct aaaagagagg gcggattccc tggccaagga gatccagtac ctgaaagatt
     1141 tgatagaaga ggtccgcaag gcaaggggga agaaaagggt cccctaccca actttcttgt
     1201 acaaagtggt atacccatac gatgttccag attacgctta gtaatgagaa atactcagaa
     1261 gatgtcactc accatagtcc gcagtgaaag ataaatgatc agaggagcat acgacttcgg
     1321 gttttagagc tagaaatagc aagtaggcca ctgagcaaga actatgaatg agatctttag
     1381 acatattgcg gccgctttct ccgcgatcca gacatgataa gatacattga tgagtttgga
     1441 caaaccaaaa ctagaatgca gtgaaaaaaa tgccttattt gtgaaatttg tgatgctatt
     1501 gccttatttg taaccattat aagctgcaat aaacaagtta tctataacaa gaaaatatat
     1561 atataataag ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta
     1621 aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag
     1681 ttcaaaatca gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg
     1741 actgagatgt cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat
     1801 ttcaagaatg catgcgtcaa ttttacgcag actatctttc tagggttaag aattcactgg
     1861 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     1921 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     1981 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
     2041 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
     2101 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2161 tgctcccggc atccgcttac agacaagctg tgaccatgtc tccgggagct gcatgtgtca
     2221 gaggttttca ccgtcatcac cgaaacacgc gagttcgaaa gggcctcgtg atacgcctat
     2281 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     2341 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2401 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     2461 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2521 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2581 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2641 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     2701 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     2761 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     2821 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     2881 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     2941 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     3001 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3061 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3121 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggt tcacgcggta
     3181 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3241 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3301 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     3361 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3421 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3481 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3541 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3601 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     3661 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3721 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3781 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3841 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3901 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3961 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4021 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4081 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4141 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4201 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gatgagcgcc
     4261 caatacgcaa accgcctctc accgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     4321 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaaa ccgcctctcc ccgcgcgttg
     4381 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     4441 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     4501 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     4561 tgaccatgat tacgccaagg tcgacttaac cctagaaaga taatcatatt gtgacgtacg
     4621 ttaaagataa tcatgcgtaa aattgacgca tgtgttttat cggtctgtat atcgaggttt
     4681 atttattaat ttgaatagat attaagtttt attatattta cacttacata ctaataataa
     4741 attcaacaaa caatttattt atgtttattt atttattaaa aaaaaacaaa aactcaaaat
     4801 ttcttctata aagtaacaaa acttttagca gtgaaaaaaa tgctttattt gtgaaatttg
     4861 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     4921 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     4981 aaacctctac aaatgtggta tggctgatta tgatcctctg gagattcatg cgccaggttt
     5041 tctggtcatg caccacgttc gtgggccctc aggaacctcc acgtcagcgg taactgtgaa
     5101 gcccagtcgc tcgtaaaaag gaagattccg gggagcactg gtttcgagga acgccggcac
     5161 gccagcccgt tccgcggcct caacaccagg aagaacgaca gcgcttccca aacctttacc
     5221 ttggtgatct gggctgacac cgacagtggc aagaaaccac gcaggctcct ttggtctatg
     5281 aggagccagc aagccctcca tttgctgttg tgccgccaac ctactccctg acagctcggc
     5341 catccgcgga cctatttccg cgaacactgc gccggcttcg acgctttccg gagtagtcca
     5401 gaccgcaact gcggcaccat cgtccgcaac ccaaacttta ccaatgtcca gacccacgcg
     5461 cgtaagaaac agctcttgca actcagttac cctctctatg tgtctgtccg gatctacggt
     5521 atggcgggtc gctggataat ctgcaaatgc tgcggcaagt gttcggacag cccgtggtac
     5581 atcgtcacgg gtagcgagcc taacagtggg cttatattcc gtcatggggc cggggttctc
     5641 ctccacgtcg ccggcctgct tcagcaggct gaagttggtg gcgccgctgc ccccggggag
     5701 catgtcaagg tcaaaatcgt caagagcgtc agcaggcagc atatcaaggt caaagtcgtc
     5761 aagggcatcg gctgggagca tgtctaagtc aaaatcgtca agggcgtcgg tcggcccgcc
     5821 gctttcgcac tttagctgtt tctccaggcc acatatgatt agttccaggc cgaaaaggaa
     5881 ggcaggttcg gctccctgcc ggtcgaacag ctcaattgct tgtttcagaa gtgggggcat
     5941 agaatcggtg gtaggtgtct ctctttcctc ttttgctact tgatgctcct gttcctccaa
     6001 tacgcagccc agtgtaaagt ggcccacggc ggacagagcg tacagtgcgt tctccaggga
     6061 gaagccttgc tgacacagga acgcgagctg attttccagg gtttcgtact gtttctctgt
     6121 tgggcgggtg ccgagatgca ctttagcccc gtcacgatgt gagaggagag cacagcggta
     6181 tgacttggcg ttgttccgca gaaagtcttg ccatgactcg ccttccaggg ggcaggagtg
     6241 ggtatgatgc ctgtccagca tctcgattgg cagggcatcg agcagggccc gcttgttctt
     6301 cacgtgccag tacagggtag gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt
     6361 caggccttcg ataccgactc cattgagtaa ttccagagca gagtttatga ctttgctctt
     6421 gtccagtcta gacatcttat cgtcatcgtc tttgtaatcc atggtggcgg atcccgcgtc
     6481 acgacacctg tgttctggcg gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg
     6541 cacttatata cggttctccc ccaccctcgg gaaaaaggcg gagccagtac acgacatcac
     6601 tttcccagtt taccccgcgc caccttctct aggcaccggt tcaattgccg acccctcccc
     6661 ccaacttctc gggcactgtg ggccatgtgc gctctgcccc atggtaatag ccatgactaa
     6721 tacgtagatg tactgccaag taggaaagtg ccataaggtc atgtactggg cataatgcca
     6781 ggcgggccat ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg
     6841 atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt
     6901 gcctattggc gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt
     6961 gggcggtcag ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg
     7021 gctatgaact aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca
     7081 taagctctaa tcgcataatc gagctaagag cgattaatat gcttagctag cttagctaag
     7141 ctagatttct ctattctccc acaatatata gcagctatta gcttagcagt ccgaatacga
     7201 tcgtccacga tccataagcg taacacattt gagtagcgac tgacgggcac cggagccact
     7261 cgagtggaat tatcacctcg agtttactcc ctatcagtga tagagaacgt atgaagagtt
     7321 tactccctat cagtgataga gaacgtatgc agactttact ccctatcagt gatagagaac
     7381 gtataaggag tttactccct atcagtgata gagaacgtat gaccagttta ctccctatca
     7441 gtgatagaga acgtatctac agtttactcc ctatcagtga tagagaacgt atatccagtt
     7501 tactccctat cagtgataga gaacgtataa g
//
