LOCUS       sequence-423608-        7062 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gene expression by Piggybac transposase in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7062)
  TITLE     Yoshimatsu Lab plasmids 2023
REFERENCE   2  (bases 1 to 7062)
  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"
COMMENT     Sequence Label: pPB-CAG-rtTAM2-IB
FEATURES             Location/Qualifiers
     source          1..7062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   660..694
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        972..1355
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1360..1637
                     /label=chicken beta-actin promoter
     intron          1638..2654
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2578..2600
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2662..2681
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(2721..3338,3339..3344,3345..3380,3381..3383,3384..3419,
                     3420..3422,3423..3458,3459..3467)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    join(3483..4057,4058..4060,4061..4069)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3650..3667)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3877..3896
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4070..4468
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4478..4599
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4515..4534)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4569..4588
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4788..4850
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(4874..4892)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4913..4929)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4913..4929)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4926..4948)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4937..4953
                     /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(4961..4967,4968..4985,4986..4991))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5006..5027
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5144..5161)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5315..5903)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5395..5414)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6074..6865,6866..6934))
                     /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     6697..6716
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6935..7039)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccttaac
      661 cctagaaaga tagtctgcgt aaaattgacg catgcattct tgaaatattg ctctctcttt
      721 ctaaatagcg cgaatccgtc gctgtgcatt taggacatct cagtcgccgc ttggagctcc
      781 cgtgaggcgt gcttgtcaat gcggtaagtg tcactgattt tgaactataa cgaccgcgtg
      841 agtcaaaatg acgcatgatt atcttttacg tgacttttaa gatttaactc atacgataat
      901 tatattgtta tttcatgttc tacttacgtg ataacttatt atatatatat tttcttgtta
      961 tagatatcgt cgacattgat tattgactag ctagttatta atagtaatca attacggggt
     1021 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     1081 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     1141 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     1201 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     1261 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     1321 agtacatcta cgtattagtc atcgctatta ccatgatggt cgaggtgagc cccacgttct
     1381 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt
     1441 aattattttg tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg
     1501 ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg
     1561 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
     1621 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
     1681 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     1741 gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt
     1801 ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
     1861 gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc
     1921 ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg
     1981 aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag
     2041 gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc
     2101 tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg
     2161 ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg
     2221 gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg
     2281 gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt
     2341 aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg
     2401 ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga
     2461 aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc
     2521 agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt
     2581 tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct
     2641 tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca
     2701 aagaattcct cgaggccacc atgtctagac tggacaagag caaagtcata aacggcgctc
     2761 tggaattact caatggagtc ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc
     2821 tgggagttga gcagcctacc ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg
     2881 ccctgccaat cgagatgctg gacaggcatc atacccactt ctgccccctg gaaggcgagt
     2941 catggcaaga ctttctgcgg aacaacgcca agtcattccg ctgtgctctc ctctcacatc
     3001 gcgacggggc taaagtgcat ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg
     3061 aaaatcagct cgcgttcctg tgtcagcaag gcttctccct ggagaacgca ctgtacgctc
     3121 tgtccgccgt gggccacttt acactgggct gcgtattgga ggaacaggag catcaagtag
     3181 caaaagagga aagagagaca cctaccaccg attctatgcc cccacttctg agacaagcaa
     3241 ttgagctgtt cgaccggcag ggagccgaac ctgccttcct tttcggcctg gaactaatca
     3301 tatgtggcct ggagaaacag ctaaagtgcg aaagcggcgg gccggccgac gcccttgacg
     3361 attttgactt agacatgctc ccagccgatg cccttgacga ctttgacctt gatatgctgc
     3421 ctgctgacgc tcttgacgat tttgaccttg acatgctccc cgggtaagcg gccgcggatc
     3481 cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg
     3541 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc
     3601 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa
     3661 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga
     3721 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc
     3781 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc
     3841 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac
     3901 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg
     3961 tacacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac
     4021 ggggacgtgg ttttcctttg aaaaacacga tgataatatg gccacaacca tggccaagcc
     4081 tttgtctcaa gaagaatcca ccctcattga aagagcaacg gctacaatca acagcatccc
     4141 catctctgaa gactacagcg tcgccagcgc agctctctct agcgacggcc gcatcttcac
     4201 tggtgtcaat gtatatcatt ttactggggg accttgtgca gaactcgtgg tgctgggcac
     4261 tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag
     4321 gggcatcttg agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat
     4381 caaagccata gtgaaggaca gtgatggaca gccgacggca gttgggattc gtgaattgct
     4441 gccctctggt tatgtgtggg agggctaatc tagagttaac ttgtttattg cagcttataa
     4501 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4561 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa cgctcgagtg catggatcct
     4621 ttgttacttt atagaagaaa ttttgagttt ttgttttttt ttaataaata aataaacata
     4681 aataaattgt ttgttgaatt tattattagt atgtaagtgt aaatataata aaacttaata
     4741 tctattcaaa ttaataaata aacctcgata tacagaccga taaaacacat gcgtcaattt
     4801 tacgcatgat tatctttaac gtacgtcaca atatgattat ctttctaggg ttaaagagct
     4861 ccagcttttg ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc
     4921 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4981 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5041 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5101 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5161 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5221 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5281 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5341 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5401 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5461 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5521 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5581 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5641 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5701 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5761 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5821 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5881 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5941 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6001 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6061 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6121 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6181 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6241 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6301 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6361 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6421 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6481 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6541 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6601 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6661 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6721 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6781 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6841 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6901 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6961 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7021 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc ac
//
