LOCUS       sequence-377032-        7258 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            KLF6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7258)
  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 7258)
  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-KLF6
FEATURES             Location/Qualifiers
     source          1..7258
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     23..47
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    113..137
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(918..942)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             944..970
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1137..1271
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1543..1577
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1588..1605)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1588..1604)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1597..1619)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1813..1832)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1934..1956
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1995..2013)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2081..2185
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2186..2254,2255..3046)
                     /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(2404..2423)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3217..3805
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3706..3725
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3959..3976
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4182..4203
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4218..4223,4224..4241,4242..4248)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4256..4272
                     /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     4261..4283
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4280..4296
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4280..4296
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4322..4384
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4560..4641
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5358..5414)
                     /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(5424..6167)
                     /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(6168..6191)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6191..6200
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6220..6431)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6432..6811
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7020..7038
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7056..7074
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7092..7110
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7128..7146
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7164..7182
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7200..7218
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7236..7254
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 ataagcttgc ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc
       61 atccacgctg ttttgacctc catagaagac accgggaccg atccagccta ccacaagttt
      121 gtacaaaaaa gttggcatgg acgtgctccc catgtgcagc atcttccagg agctccagat
      181 cgtgcacgag accggctact tctcggcgct gccgtctctg gaggagtact ggcaacagac
      241 ctgcctagag ctggaacgtt acctccagag cgagccctgc tatgtttcag cctcagaaat
      301 caaatttgac agccaggaag atctgtggac caaaatcatt ctggctcggg agaaaaagga
      361 ggaatccgaa ctgaagatat cttccagtcc tccagaggac actctcatca gcccgagctt
      421 ttgttacaac ttagagacca acagcctgaa ctcagatgtc agcagcgaat cctctgacag
      481 ctccgaggaa ctttctccca cggccaagtt tacctccgac cccattggcg aagttttggt
      541 cagctcggga aaattgagct cctctgtcac ctccacgcct ccatcttctc cggaactgag
      601 cagggaacct tctcaactgt ggggttgcgt gcccggggag ctgccctcgc cagggaaggt
      661 gcgcagcggg acttcgggga agccaggtga caagggaaat ggcgatgcct cccccgacgg
      721 caggaggagg gtgcaccggt gccactttaa cggctgcagg aaagtttaca ccaaaagctc
      781 ccacttgaaa gcacaccagc ggacgcacac aggagaaaag ccttacagat gctcatggga
      841 agggtgtgag tggcgttttg caagaagtga tgagttaacc aggcacttcc gaaagcacac
      901 cggggccaag ccttattacc caactttctt gtacaaagtg gtatacccat acgatgttcc
      961 agattacgct tagtaatgag aaatactcag aagatgtcac tcaccatagt ccgcagtgaa
     1021 agataaatga tcctgtaaat tgtgttgtgc cggttttaga gctagaaata gcaagtaggc
     1081 cactgagcaa gaactatgaa tgagatcttt agacatattg cggccgcttt ctccgcgatc
     1141 cagacatgat aagatacatt gatgagtttg gacaaaccaa aactagaatg cagtgaaaaa
     1201 aatgccttat ttgtgaaatt tgtgatgcta ttgccttatt tgtaaccatt ataagctgca
     1261 ataaacaagt tatctataac aagaaaatat atatataata agttatcacg taagtagaac
     1321 atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc
     1381 atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg
     1441 acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg
     1501 gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc
     1561 agactatctt tctagggtta agaattcact ggccgtcgtt ttacaacgtc gtgactggga
     1621 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     1681 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     1741 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     1801 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     1861 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     1921 tgtgaccatg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacac
     1981 gcgagttcga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     2041 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2101 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2161 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2221 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     2281 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     2341 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     2401 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     2461 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2521 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2581 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     2641 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     2701 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     2761 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2821 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2881 atctggagcc ggtgagcgtg gttcacgcgg tatcattgca gcactggggc cagatggtaa
     2941 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3001 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3061 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3121 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3181 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3241 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3301 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3361 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3421 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3481 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3541 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3601 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3661 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3721 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3781 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3841 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3901 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3961 cgagtcagtg agcgaggaag cggatgagcg cccaatacgc aaaccgcctc tcaccgcgcg
     4021 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     4081 gcgcaacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     4141 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     4201 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     4261 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa ggtcgactta
     4321 accctagaaa gataatcata ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg
     4381 catgtgtttt atcggtctgt atatcgaggt ttatttatta atttgaatag atattaagtt
     4441 ttattatatt tacacttaca tactaataat aaattcaaca aacaatttat ttatgtttat
     4501 ttatttatta aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca aaacttttag
     4561 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     4621 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     4681 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat
     4741 tatgatcctc tggagattca tgcgccaggt tttctggtca tgcaccacgt tcgtgggccc
     4801 tcaggaacct ccacgtcagc ggtaactgtg aagcccagtc gctcgtaaaa aggaagattc
     4861 cggggagcac tggtttcgag gaacgccggc acgccagccc gttccgcggc ctcaacacca
     4921 ggaagaacga cagcgcttcc caaaccttta ccttggtgat ctgggctgac accgacagtg
     4981 gcaagaaacc acgcaggctc ctttggtcta tgaggagcca gcaagccctc catttgctgt
     5041 tgtgccgcca acctactccc tgacagctcg gccatccgcg gacctatttc cgcgaacact
     5101 gcgccggctt cgacgctttc cggagtagtc cagaccgcaa ctgcggcacc atcgtccgca
     5161 acccaaactt taccaatgtc cagacccacg cgcgtaagaa acagctcttg caactcagtt
     5221 accctctcta tgtgtctgtc cggatctacg gtatggcggg tcgctggata atctgcaaat
     5281 gctgcggcaa gtgttcggac agcccgtggt acatcgtcac gggtagcgag cctaacagtg
     5341 ggcttatatt ccgtcatggg gccggggttc tcctccacgt cgccggcctg cttcagcagg
     5401 ctgaagttgg tggcgccgct gcccccgggg agcatgtcaa ggtcaaaatc gtcaagagcg
     5461 tcagcaggca gcatatcaag gtcaaagtcg tcaagggcat cggctgggag catgtctaag
     5521 tcaaaatcgt caagggcgtc ggtcggcccg ccgctttcgc actttagctg tttctccagg
     5581 ccacatatga ttagttccag gccgaaaagg aaggcaggtt cggctccctg ccggtcgaac
     5641 agctcaattg cttgtttcag aagtgggggc atagaatcgg tggtaggtgt ctctctttcc
     5701 tcttttgcta cttgatgctc ctgttcctcc aatacgcagc ccagtgtaaa gtggcccacg
     5761 gcggacagag cgtacagtgc gttctccagg gagaagcctt gctgacacag gaacgcgagc
     5821 tgattttcca gggtttcgta ctgtttctct gttgggcggg tgccgagatg cactttagcc
     5881 ccgtcacgat gtgagaggag agcacagcgg tatgacttgg cgttgttccg cagaaagtct
     5941 tgccatgact cgccttccag ggggcaggag tgggtatgat gcctgtccag catctcgatt
     6001 ggcagggcat cgagcagggc ccgcttgttc ttcacgtgcc agtacagggt aggctgctca
     6061 actcccagct tttgagcgag tttccttgtc gtcaggcctt cgataccgac tccattgagt
     6121 aattccagag cagagtttat gactttgctc ttgtccagtc tagacatctt atcgtcatcg
     6181 tctttgtaat ccatggtggc ggatcccgcg tcacgacacc tgtgttctgg cggcaaaccc
     6241 gttgcgaaaa agaacgttca cggcgactac tgcacttata tacggttctc ccccaccctc
     6301 gggaaaaagg cggagccagt acacgacatc actttcccag tttaccccgc gccaccttct
     6361 ctaggcaccg gttcaattgc cgacccctcc ccccaacttc tcgggcactg tgggccatgt
     6421 gcgctctgcc ccatggtaat agccatgact aatacgtaga tgtactgcca agtaggaaag
     6481 tgccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca
     6541 atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt
     6601 aaatactcca cccattgacg tcaatggaaa gtgcctattg gcgttactat gggaacatac
     6661 gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc
     6721 gtaagttatg taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat
     6781 tactattaat aactagtcaa taatcaatgt cataagctct aatcgcataa tcgagctaag
     6841 agcgattaat atgcttagct agcttagcta agctagattt ctctattctc ccacaatata
     6901 tagcagctat tagcttagca gtccgaatac gatcgtccac gatccataag cgtaacacat
     6961 ttgagtagcg actgacgggc accggagcca ctcgagtgga attatcacct cgagtttact
     7021 ccctatcagt gatagagaac gtatgaagag tttactccct atcagtgata gagaacgtat
     7081 gcagacttta ctccctatca gtgatagaga acgtataagg agtttactcc ctatcagtga
     7141 tagagaacgt atgaccagtt tactccctat cagtgataga gaacgtatct acagtttact
     7201 ccctatcagt gatagagaac gtatatccag tttactccct atcagtgata gagaacgt
//
