LOCUS       sequence-377017-        7258 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Barcoded piggybac transposon vector with Dox-inducible expression of
            HOXC9.
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 Apr 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-HOXC9
FEATURES             Location/Qualifiers
     source          1..7258
                     /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(916..940)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             942..968
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    1135..1269
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1541..1575
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1586..1603)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1586..1602)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1595..1617)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1811..1830)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1932..1954
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1993..2011)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2079..2183
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2184..2252,2253..3044)
                     /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(2402..2421)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3215..3803
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3704..3723
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3957..3974
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4180..4201
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4216..4221,4222..4239,4240..4246)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4254..4270
                     /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     4259..4281
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4278..4294
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4278..4294
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4320..4382
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4558..4639
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5356..5412)
                     /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(5422..6165)
                     /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(6166..6189)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6189..6198
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6218..6429)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6430..6809
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    7018..7036
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7054..7072
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7090..7108
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7126..7144
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7162..7180
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7198..7216
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7234..7252
                     /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 tggcatgtcg gcgacggggc ccatcagtaa ctattacgtg gactcgctca
      181 tctctcacga caatgaagac ctcctagcgt ccaggtttcc ggccaccggg gctcatcccg
      241 ccgccgccag acccagcggt ttggtgccgg actgtagcga ttttccgtcc tgtagcttcg
      301 cgcccaagcc ggcagtgttc agcacgtcgt gggcgcccgt gccctctcag tcgtccgtgg
      361 tatatcaccc gtacggcccc cagccccacc tcggcgccga cacgcgctac atgcggactt
      421 ggctcgagcc gctgtccggc gccgtctcct tccccagctt cccggccggg ggccgtcact
      481 acgccctcaa gccggacgcc taccccgggc gccgcgcgga ctgcggccca ggggagggcc
      541 gcagctaccc ggactacatg tacggctcgc ccggggagct gcgcgaccgc gccccgcaga
      601 cactgccctc gcccgaggcg gacgcgctcg ccggcagcaa gcacaaagag gagaaggctg
      661 acctggaccc cagcaacccc gtggccaact ggattcacgc ccgctccacg aggaagaagc
      721 gctgccccta caccaagtac cagacgctgg aactggagaa ggagtttctc ttcaatatgt
      781 atttaaccag ggaccgtcgg tatgaggtgg cccgggttct caatctcacc gagcggcagg
      841 tcaaaatctg gtttcagaat cgaaggatga agatgaaaaa gatgaataaa gagaaaaccg
      901 acaaggagca gtcctaccca actttcttgt acaaagtggt atacccatac gatgttccag
      961 attacgctta gtaatgagaa atactcagaa gatgtcactc accatagtcc gcagtgaaag
     1021 ataaatgatc gacccaaatt tcaaaagaat gttttagagc tagaaatagc aagtaggcca
     1081 ctgagcaaga actatgaatg agatctttag acatattgcg gccgctttct ccgcgatcca
     1141 gacatgataa gatacattga tgagtttgga caaaccaaaa ctagaatgca gtgaaaaaaa
     1201 tgccttattt gtgaaatttg tgatgctatt gccttatttg taaccattat aagctgcaat
     1261 aaacaagtta tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat
     1321 gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat
     1381 gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac
     1441 aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga
     1501 ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag
     1561 actatctttc tagggttaag aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     1621 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     1681 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     1741 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     1801 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     1861 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     1921 tgaccatgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacacgc
     1981 gagttcgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     2041 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2101 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2161 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2221 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2281 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2341 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2401 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     2461 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2521 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     2581 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     2641 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     2701 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     2761 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     2821 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     2881 ctggagccgg tgagcgtggt tcacgcggta tcattgcagc actggggcca gatggtaagc
     2941 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3001 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3061 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3121 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3181 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3241 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3301 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3361 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3421 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3481 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3541 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3601 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3661 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3721 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3781 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3841 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3901 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3961 agtcagtgag cgaggaagcg gatgagcgcc caatacgcaa accgcctctc accgcgcgtt
     4021 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     4081 gcaacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     4141 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     4201 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     4261 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagg tcgacttaac
     4321 cctagaaaga taatcatatt gtgacgtacg ttaaagataa tcatgcgtaa aattgacgca
     4381 tgtgttttat cggtctgtat atcgaggttt atttattaat ttgaatagat attaagtttt
     4441 attatattta cacttacata ctaataataa attcaacaaa caatttattt atgtttattt
     4501 atttattaaa aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa acttttagca
     4561 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     4621 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     4681 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
     4741 tgatcctctg gagattcatg cgccaggttt tctggtcatg caccacgttc gtgggccctc
     4801 aggaacctcc acgtcagcgg taactgtgaa gcccagtcgc tcgtaaaaag gaagattccg
     4861 gggagcactg gtttcgagga acgccggcac gccagcccgt tccgcggcct caacaccagg
     4921 aagaacgaca gcgcttccca aacctttacc ttggtgatct gggctgacac cgacagtggc
     4981 aagaaaccac gcaggctcct ttggtctatg aggagccagc aagccctcca tttgctgttg
     5041 tgccgccaac ctactccctg acagctcggc catccgcgga cctatttccg cgaacactgc
     5101 gccggcttcg acgctttccg gagtagtcca gaccgcaact gcggcaccat cgtccgcaac
     5161 ccaaacttta ccaatgtcca gacccacgcg cgtaagaaac agctcttgca actcagttac
     5221 cctctctatg tgtctgtccg gatctacggt atggcgggtc gctggataat ctgcaaatgc
     5281 tgcggcaagt gttcggacag cccgtggtac atcgtcacgg gtagcgagcc taacagtggg
     5341 cttatattcc gtcatggggc cggggttctc ctccacgtcg ccggcctgct tcagcaggct
     5401 gaagttggtg gcgccgctgc ccccggggag catgtcaagg tcaaaatcgt caagagcgtc
     5461 agcaggcagc atatcaaggt caaagtcgtc aagggcatcg gctgggagca tgtctaagtc
     5521 aaaatcgtca agggcgtcgg tcggcccgcc gctttcgcac tttagctgtt tctccaggcc
     5581 acatatgatt agttccaggc cgaaaaggaa ggcaggttcg gctccctgcc ggtcgaacag
     5641 ctcaattgct tgtttcagaa gtgggggcat agaatcggtg gtaggtgtct ctctttcctc
     5701 ttttgctact tgatgctcct gttcctccaa tacgcagccc agtgtaaagt ggcccacggc
     5761 ggacagagcg tacagtgcgt tctccaggga gaagccttgc tgacacagga acgcgagctg
     5821 attttccagg gtttcgtact gtttctctgt tgggcgggtg ccgagatgca ctttagcccc
     5881 gtcacgatgt gagaggagag cacagcggta tgacttggcg ttgttccgca gaaagtcttg
     5941 ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc ctgtccagca tctcgattgg
     6001 cagggcatcg agcagggccc gcttgttctt cacgtgccag tacagggtag gctgctcaac
     6061 tcccagcttt tgagcgagtt tccttgtcgt caggccttcg ataccgactc cattgagtaa
     6121 ttccagagca gagtttatga ctttgctctt gtccagtcta gacatcttat cgtcatcgtc
     6181 tttgtaatcc atggtggcgg atcccgcgtc acgacacctg tgttctggcg gcaaacccgt
     6241 tgcgaaaaag aacgttcacg gcgactactg cacttatata cggttctccc ccaccctcgg
     6301 gaaaaaggcg gagccagtac acgacatcac tttcccagtt taccccgcgc caccttctct
     6361 aggcaccggt tcaattgccg acccctcccc ccaacttctc gggcactgtg ggccatgtgc
     6421 gctctgcccc atggtaatag ccatgactaa tacgtagatg tactgccaag taggaaagtg
     6481 ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat
     6541 agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa
     6601 atactccacc cattgacgtc aatggaaagt gcctattggc gttactatgg gaacatacgt
     6661 cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt
     6721 aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta
     6781 ctattaataa ctagtcaata atcaatgtca taagctctaa tcgcataatc gagctaagag
     6841 cgattaatat gcttagctag cttagctaag ctagatttct ctattctccc acaatatata
     6901 gcagctatta gcttagcagt ccgaatacga tcgtccacga tccataagcg taacacattt
     6961 gagtagcgac tgacgggcac cggagccact cgagtggaat tatcacctcg agtttactcc
     7021 ctatcagtga tagagaacgt atgaagagtt tactccctat cagtgataga gaacgtatgc
     7081 agactttact ccctatcagt gatagagaac gtataaggag tttactccct atcagtgata
     7141 gagaacgtat gaccagttta ctccctatca gtgatagaga acgtatctac agtttactcc
     7201 ctatcagtga tagagaacgt atatccagtt tactccctat cagtgataga gaacgtat
//
