LOCUS       sequence-173247-        6620 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  PiggyBac backbone, CAG promoter, Hygromycin selection, N-terminal 
            3xFLAG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6620)
  AUTHORS   Betschinger J, Nichols J, Dietmann S, Corrin PD, Paddison PJ, Smith 
            A
  TITLE     Exit from pluripotency is gated by intracellular redistribution of 
            the bHLH transcription factor Tfe3.
  JOURNAL   Cell. 2013 Apr 11;153(2):335-47. doi: 10.1016/j.cell.2013.03.012.
  PUBMED    23582324
REFERENCE   2  (bases 1 to 6620)
  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"; doi: 
            "10.1016/j.cell.2013.03"; journalName: "Cell"; date: "2013-04-11- 
            11"; volume: "153"; issue: "2"; pages: "335-47"
COMMENT     Sequence Label: pPB-CAG-3xFLAG-empty-pgk-hph
FEATURES             Location/Qualifiers
     source          1..6620
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     142..164
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     226..245
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             297..362
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(399..415)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    423..439
                     /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)."
     promoter        complement(join(447..453,454..471,472..477))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    492..513
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(573..590)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    579..803
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    880..913
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     repeat_region   1084..1118
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1136..1155)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1137..1155)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1162..1179)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1162..1178)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1171..1193)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1320..1775
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1407..1426)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1617..1638
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1801..1905
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1906..1974,1975..2766)
                     /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(2124..2143)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2937..3525
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3426..3445
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3679..3696
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3813..3834
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3849..3854,3855..3872,3873..3879)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3887..3903
                     /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     3911..3927
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3948..3966
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   3989..4023
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     CDS             complement(4505..5530)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(4505..5530)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(5549..6048)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5597..5616)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     5990..6013
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    6071..6104
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        6120..6499
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
       61 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
      121 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
      181 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
      241 ttgtgctgtc tcatcatttt ggcaaagaat tcctcgacgg tatcgataag cttatggact
      301 acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat gacgatgaca
      361 agggatcccc cggggtcgac aagcttggcg taatcatggt catagctgtt tcctgtgtga
      421 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
      481 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
      541 cagtcgggaa acctgtcgtg ccagcggatc agcctcgact gtgccttcta gttgccagcc
      601 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
      661 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
      721 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
      781 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgagatc
      841 cactagttct agcctcgagg ctagagcggc cgcgtcgaga taacttcgta taatgtatgc
      901 tatacgaagt tatgttttgt tactttatag aagaaatttt gagtttttgt ttttttttaa
      961 taaataaata aacataaata aattgtttgt tgaatttatt attagtatgt aagtgtaaat
     1021 ataataaaac ttaatatcta ttcaaattaa taaataaacc tcgatataca gaccgataaa
     1081 acacatgcgt caattttacg cagaytatct ttctagggtt aaggtaccca attcgcccta
     1141 tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     1201 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     1261 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     1321 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1381 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1441 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1501 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     1561 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     1621 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     1681 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     1741 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     1801 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     1861 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     1921 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     1981 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     2041 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     2101 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     2161 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     2221 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     2281 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     2341 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     2401 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     2461 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     2521 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     2581 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     2641 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     2701 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     2761 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     2821 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     2881 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     2941 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3001 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3061 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     3121 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3181 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3241 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3301 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3361 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3421 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3481 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3541 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3601 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3661 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     3721 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     3781 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     3841 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     3901 caatttcaca caggaaacag ctatgaccat gattacgcca agctcgaaat taaccctcac
     3961 taaagggmgm awnanntggg taccttaacc ctagaaagat agtctgcgta aaattgacgc
     4021 atgcattctt gaaatattgc tctctctttc taaatagcgc gaatccgtcg ctgtgcattt
     4081 aggacatctc agtcgccgct tggagctccc gtgaggcgtg cttgtcaatg cggtaagtgt
     4141 cactgatttt gaactataac gaccgcgtga gtcaaaatga cgcatgatta tcttttacgt
     4201 gacttttaag atttaactca tacgataatt atattgttat ttcatgttct acttacgtga
     4261 taacttatta tatatatatt ttcttgttat agatatccaa ctatgaaaca ttatcataaa
     4321 gcaatagtaa agagccttca gtaaagagca ggcatttatc taatcccacc ccacccccac
     4381 ccccgtagct ccaatccttc cattcaaaat gtaggtactc tgttctcacc cttcttaaca
     4441 aagtatgaca ggaaaaactt ccatgtggac atctttattg tttaatagat catcaatttc
     4501 tcgactattc ctttgccctc ggacgagtgc tggggcgtcg gtttccacta tcggcgagta
     4561 cttctacaca gccatcggtc cagacggccg cgcttctgcg ggcgatttgt gtacgcccga
     4621 cagtcccggc tccggatcgg acgattgcgt cgcatcgacc ctgcgcccaa gctgcatcat
     4681 cgaaattgcc gtcaaccaag ctctgataga gttggtcaag accaatgcgg agcatatacg
     4741 cccggagccg cggcgatcct gcaagctccg gatgcctccg ctcgaagtag cgcgtctgct
     4801 gctccataca agccaaccac ggcctccaga agaagatgtt ggcgacctcg tattgggaat
     4861 ccccgaacat cgcctcgctc cagtcaatga ccgctgttat gcggccattg tccgtcagga
     4921 cattgttgga gccgaaatcc gcgtgcacga ggtgccggac ttcggggcag tcctcggccc
     4981 aaagcatcag ctcatcgaga gcctgcgcga cggacgcact gacggtgtcg tccatcacag
     5041 tttgccagtg atacacatgg ggatcagcaa tcgcgcatat gaaatcacgc catgtagtgt
     5101 attgaccgat tccttgcggt ccgaatgggc cgaacccgct cgtctggcta agatcggccg
     5161 cagcgatcgc atccatggcc tccgcgaccg gctgcagaac agcgggcagt tcggtttcag
     5221 gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat gcaataggtc aggctctcgc
     5281 tgaattcccc aatgtcaagc acttccggaa tcgggagcgc ggccgatgca aagtgccgat
     5341 aaacataacg atctttgtag aaaccatcgg cgcagctatt tacccgcagg acatatccac
     5401 gccctcctac atcgaagctg aaagcacgag attcttcgcc ctccgagagc tgcatcaggt
     5461 cggagacgct gtcgaacttt tcgatcagaa acttctcgac agacgtcgcg gtgagttcag
     5521 gctttttcat ggtggcggct ggatcggtcg aaaggcccgg agatgaggaa gaggagaaca
     5581 gcgcggcaga cgtgcgcttt tgaagcgtgc agaatgccgg gcctccggag gaccttcggg
     5641 cgcccgcccc gcccctgagc ccgcccctga gcccgccccc ggacccaccc cttcccagcc
     5701 tctgagccca gaaagcgaag gagcaaagct gctattggcc gctgccccaa aggcctaccc
     5761 gcttccattg ctcagcggtg ctgtccatct gcacgagact agtgagacgt gctacttcca
     5821 tttgtcacgt cctgcacgac gcgagctgcg gggcgggggg gaacttcctg actaggggag
     5881 gagtagaagg tggcgcgaag gggccaccaa agaacggagc cggttggcgc ctaccggtgg
     5941 atgtggaatg tgtgcgaggc cagaggccac ttgtgtagcg ccaagtgccc agcggggctg
     6001 ctaaagcgca tgctccagac tgccttggga aaagcgcctc ccctacccgg tagaattcat
     6061 cgatgatatc ataacttcgt ataatgtatg ctatacgaag ttatgcggcc ctcgaggtcg
     6121 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     6181 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     6241 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     6301 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     6361 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     6421 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     6481 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc
     6541 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat
     6601 gggggcgggg gggggggggg
//
