LOCUS       sequence-348266-        6493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one piggyBac transposon vector for dox-inducible expression 
            of Golden Gate cloned elements (CMVe-hEF1a-driven rtTA and puromycin
            resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6493)
  AUTHORS   Kramme C, Plesa AM, Wang HH, Wolf B, Smela MP, Guo X, Kohman RE, 
            Chatterjee P, Church GM
  TITLE     An integrated pipeline for mammalian genetic screening.
  JOURNAL   Cell Rep Methods. 2021 Sep 27;1(6):100082. doi: 
            10.1016/j.crmeth.2021.100082. eCollection 2021 Oct 25.
  PUBMED    35474898
REFERENCE   2  (bases 1 to 6493)
  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: "Cell Rep 
            Methods."; date: "2021-09-27"; volume: "1(6):100082. doi"; pages: " 
            10.1016/j.crmeth.2021.100082. eCollection 2021 Oct 25"
COMMENT     Sequence Label: PB-T3G-CERP2
FEATURES             Location/Qualifiers
     source          1..6493
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        8..375
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    16..34
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    52..70
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    88..106
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    124..142
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    160..178
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    196..214
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    232..250
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     polyA_signal    622..756
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1032..1066
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1077..1094)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1077..1093)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1086..1108)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1302..1321)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1423..1445
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1484..1502)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1570..1674
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1675..1743,1744..2535)
                     /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(1893..1912)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2706..3294
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3195..3214
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3448..3465
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3671..3692
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3707..3712,3713..3730,3731..3737)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3745..3761
                     /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     3750..3772
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3769..3785
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3769..3785
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   3811..3873
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4049..4130
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4847..4903)
                     /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(4913..5656)
                     /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(5657..5680)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      5680..5689
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5709..5920)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        5921..6300
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tcacctcgag tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca
       61 gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt
      121 tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac
      181 gtatctacag tttactccct atcagtgata gagaacgtat atccagttta ctccctatca
      241 gtgatagaga acgtataagc tttgcttatg taaaccaggg cgcctataaa agagtgctga
      301 ttttttgagt aaacttcaat tccacaacac ttttgtctta taccaacttt ccgtaccact
      361 tcctaccctc gtaaaggtac cgccacccga gaccattaat gggtctcgta atgataggcg
      421 tcttcgaata atggaagaca tactagtaga ccacctcccc tgcgagctaa gctggacagc
      481 caatgacggg taagagagtg acatttttca ctaacctaag acaggagggc cgtcagagct
      541 actgcctaat ccaaagacgg gtaaaagtga taaaaatgta tcactccaac ctaagacagg
      601 cgcagcttcc gagggatttg agatccagac atgataagat acattgatga gtttggacaa
      661 accaaaacta gaatgcagtg aaaaaaatgc cttatttgtg aaatttgtga tgctattgcc
      721 ttatttgtaa ccattataag ctgcaataaa caagtttgat atctataaca agaaaatata
      781 tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt
      841 aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata
      901 gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc
      961 gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata
     1021 tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa gaattcactg
     1081 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     1141 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     1201 tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg
     1261 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
     1321 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     1381 ctgctcccgg catccgctta cagacaagct gtgaccatgt ctccgggagc tgcatgtgtc
     1441 agaggttttc accgtcatca ccgaaacacg cgagttcgaa agggcctcgt gatacgccta
     1501 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     1561 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1621 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     1681 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     1741 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     1801 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     1861 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     1921 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1981 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     2041 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     2101 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     2161 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     2221 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     2281 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     2341 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg ttcacgcggt
     2401 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     2461 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     2521 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     2581 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     2641 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2701 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2761 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2821 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     2881 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     2941 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3001 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3061 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3121 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3181 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3241 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3301 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3361 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3421 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggatgagcgc
     3481 ccaatacgca aaccgcctct caccgcgcgt tggccgattc attaatgcag ctggcacgac
     3541 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa accgcctctc cccgcgcgtt
     3601 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     3661 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     3721 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     3781 atgaccatga ttacgccaag gtcgacttaa ccctagaaag ataatcatat tgtgacgtac
     3841 gttaaagata atcatgcgta aaattgacgc atgtgtttta tcggtctgta tatcgaggtt
     3901 tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata
     3961 aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa
     4021 tttcttctat aaagtaacaa aacttttagc agtgaaaaaa atgctttatt tgtgaaattt
     4081 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     4141 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     4201 aaaacctcta caaatgtggt atggctgatt atgatcctct ggagattcat gcgccaggtt
     4261 ttctggtcat gcaccacgtt cgtgggccct caggaacctc cacgtcagcg gtaactgtga
     4321 agcccagtcg ctcgtaaaaa ggaagattcc ggggagcact ggtttcgagg aacgccggca
     4381 cgccagcccg ttccgcggcc tcaacaccag gaagaacgac agcgcttccc aaacctttac
     4441 cttggtgatc tgggctgaca ccgacagtgg caagaaacca cgcaggctcc tttggtctat
     4501 gaggagccag caagccctcc atttgctgtt gtgccgccaa cctactccct gacagctcgg
     4561 ccatccgcgg acctatttcc gcgaacactg cgccggcttc gacgctttcc ggagtagtcc
     4621 agaccgcaac tgcggcacca tcgtccgcaa cccaaacttt accaatgtcc agacccacgc
     4681 gcgtaagaaa cagctcttgc aactcagtta ccctctctat gtgtctgtcc ggatctacgg
     4741 tatggcgggt cgctggataa tctgcaaatg ctgcggcaag tgttcggaca gcccgtggta
     4801 catcgtcacg ggtagcgagc ctaacagtgg gcttatattc cgtcatgggg ccggggttct
     4861 cctccacgtc gccggcctgc ttcagcaggc tgaagttggt ggcgccgctg cccccgggga
     4921 gcatgtcaag gtcaaaatcg tcaagagcgt cagcaggcag catatcaagg tcaaagtcgt
     4981 caagggcatc ggctgggagc atgtctaagt caaaatcgtc aagggcgtcg gtcggcccgc
     5041 cgctttcgca ctttagctgt ttctccaggc cacatatgat tagttccagg ccgaaaagga
     5101 aggcaggttc ggctccctgc cggtcgaaca gctcaattgc ttgtttcaga agtgggggca
     5161 tagaatcggt ggtaggtgtc tctctttcct cttttgctac ttgatgctcc tgttcctcca
     5221 atacgcagcc cagtgtaaag tggcccacgg cggacagagc gtacagtgcg ttctccaggg
     5281 agaagccttg ctgacacagg aacgcgagct gattttccag ggtttcgtac tgtttctctg
     5341 ttgggcgggt gccgagatgc actttagccc cgtcacgatg tgagaggaga gcacagcggt
     5401 atgacttggc gttgttccgc agaaagtctt gccatgactc gccttccagg gggcaggagt
     5461 gggtatgatg cctgtccagc atctcgattg gcagggcatc gagcagggcc cgcttgttct
     5521 tcacgtgcca gtacagggta ggctgctcaa ctcccagctt ttgagcgagt ttccttgtcg
     5581 tcaggccttc gataccgact ccattgagta attccagagc agagtttatg actttgctct
     5641 tgtccagtct agacatctta tcgtcatcgt ctttgtaatc catggtggcg gatcccgcgt
     5701 cacgacacct gtgttctggc ggcaaacccg ttgcgaaaaa gaacgttcac ggcgactact
     5761 gcacttatat acggttctcc cccaccctcg ggaaaaaggc ggagccagta cacgacatca
     5821 ctttcccagt ttaccccgcg ccaccttctc taggcaccgg ttcaattgcc gacccctccc
     5881 cccaacttct cgggcactgt gggccatgtg cgctctgccc catggtaata gccatgacta
     5941 atacgtagat gtactgccaa gtaggaaagt gccataaggt catgtactgg gcataatgcc
     6001 aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt
     6061 gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag
     6121 tgcctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt
     6181 tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg
     6241 ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc
     6301 ataagctcta atcgcataat cgagctaaga gcgattaata tgcttagcta gcttagctaa
     6361 gctagatttc tctattctcc cacaatatat agcagctatt agcttagcag tccgaatacg
     6421 atcgtccacg atccataagc gtaacacatt tgagtagcga ctgacgggca ccggagccac
     6481 tcgagtggaa tta
//
