LOCUS       sequence-344442-        6232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one piggyBac transposon destination vector for 
            mCMV+dox-inducible expression of Mega Gate cloned elements 
            (hEF1a-driven rtTA and puromycin resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6232)
  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 6232)
  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-CT3G-ERP2-MG
FEATURES             Location/Qualifiers
     source          1..6232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     278..302
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    386..510
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(631..755)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             757..783
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    905..1039
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1311..1345
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1356..1373)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1356..1372)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1365..1387)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1581..1600)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1702..1724
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1763..1781)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1849..1953
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1954..2022,2023..2814)
                     /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(2172..2191)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2985..3573
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3474..3493
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3727..3744
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3950..3971
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3986..3991,3992..4009,4010..4016)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4024..4040
                     /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     4029..4051
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4048..4064
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4048..4064
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4090..4152
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4328..4409
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5126..5182)
                     /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(5192..5935)
                     /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(5936..5959)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      5959..5968
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5988..6199)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag cttgcctata taagcagagc tcgtttagtg aaccgtcaga
      301 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      361 gcctccgcgg tggcggcatc ggtacacaag tttgtacaaa aaagctgaac gagaaacgta
      421 aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata
      481 ctgtaaaaca caacatatcc agtcactatg gcgacagaag aagtataggg ataacagggt
      541 aattgttgta agcgcgctat gatggaggct atgccactag aatctgcgtt cgctacctta
      601 ggaccgttat agttagaagg aaagctccat catagtgact ggatatgttg tgttttacag
      661 tattatgtag tctgtttttt atgcaaaatc taatttaata tattgatatt tatatcattt
      721 tacgtttctc gttcagcttt cttgtacaaa gtggtatacc catacgatgt tccagattac
      781 gcttagtaat gagaaatact cagaagatgt cactcaccat agagagacca ttaatgggtc
      841 tcctaggcca ctgagcaaga actatgaatg agatctttag acatattgcg gccgctttct
      901 ccgcgatcca gacatgataa gatacattga tgagtttgga caaaccaaaa ctagaatgca
      961 gtgaaaaaaa tgccttattt gtgaaatttg tgatgctatt gccttatttg taaccattat
     1021 aagctgcaat aaacaagtta tctataacaa gaaaatatat atataataag ttatcacgta
     1081 agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa
     1141 agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta
     1201 ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca
     1261 cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa
     1321 ttttacgcag actatctttc tagggttaag aattcactgg ccgtcgtttt acaacgtcgt
     1381 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     1441 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     1501 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     1561 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     1621 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     1681 agacaagctg tgaccatgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     1741 cgaaacacgc gagttcgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     1801 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     1861 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     1921 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     1981 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2041 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2101 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2161 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2221 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2281 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2341 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2401 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2461 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     2521 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2581 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2641 gctgataaat ctggagccgg tgagcgtggt tcacgcggta tcattgcagc actggggcca
     2701 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2761 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     2821 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     2881 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     2941 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3001 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3061 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3121 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3181 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3241 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3301 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3361 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3421 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3481 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3541 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3601 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3661 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3721 gagcgcagcg agtcagtgag cgaggaagcg gatgagcgcc caatacgcaa accgcctctc
     3781 accgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     3841 ggcagtgagc gcaacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     3901 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     3961 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     4021 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagg
     4081 tcgacttaac cctagaaaga taatcatatt gtgacgtacg ttaaagataa tcatgcgtaa
     4141 aattgacgca tgtgttttat cggtctgtat atcgaggttt atttattaat ttgaatagat
     4201 attaagtttt attatattta cacttacata ctaataataa attcaacaaa caatttattt
     4261 atgtttattt atttattaaa aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa
     4321 acttttagca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     4381 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     4441 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
     4501 tggctgatta tgatcctctg gagattcatg cgccaggttt tctggtcatg caccacgttc
     4561 gtgggccctc aggaacctcc acgtcagcgg taactgtgaa gcccagtcgc tcgtaaaaag
     4621 gaagattccg gggagcactg gtttcgagga acgccggcac gccagcccgt tccgcggcct
     4681 caacaccagg aagaacgaca gcgcttccca aacctttacc ttggtgatct gggctgacac
     4741 cgacagtggc aagaaaccac gcaggctcct ttggtctatg aggagccagc aagccctcca
     4801 tttgctgttg tgccgccaac ctactccctg acagctcggc catccgcgga cctatttccg
     4861 cgaacactgc gccggcttcg acgctttccg gagtagtcca gaccgcaact gcggcaccat
     4921 cgtccgcaac ccaaacttta ccaatgtcca gacccacgcg cgtaagaaac agctcttgca
     4981 actcagttac cctctctatg tgtctgtccg gatctacggt atggcgggtc gctggataat
     5041 ctgcaaatgc tgcggcaagt gttcggacag cccgtggtac atcgtcacgg gtagcgagcc
     5101 taacagtggg cttatattcc gtcatggggc cggggttctc ctccacgtcg ccggcctgct
     5161 tcagcaggct gaagttggtg gcgccgctgc ccccggggag catgtcaagg tcaaaatcgt
     5221 caagagcgtc agcaggcagc atatcaaggt caaagtcgtc aagggcatcg gctgggagca
     5281 tgtctaagtc aaaatcgtca agggcgtcgg tcggcccgcc gctttcgcac tttagctgtt
     5341 tctccaggcc acatatgatt agttccaggc cgaaaaggaa ggcaggttcg gctccctgcc
     5401 ggtcgaacag ctcaattgct tgtttcagaa gtgggggcat agaatcggtg gtaggtgtct
     5461 ctctttcctc ttttgctact tgatgctcct gttcctccaa tacgcagccc agtgtaaagt
     5521 ggcccacggc ggacagagcg tacagtgcgt tctccaggga gaagccttgc tgacacagga
     5581 acgcgagctg attttccagg gtttcgtact gtttctctgt tgggcgggtg ccgagatgca
     5641 ctttagcccc gtcgcgatgt gagaggagag cacagcggta tgacttggcg ttgttccgca
     5701 gaaagtcttg ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc ctgtccagca
     5761 tctcgattgg cagggcatcg agcagggccc gcttgttctt cacgtgccag tacagggtag
     5821 gctgctcaac tcccagcttt tgagcgagtt tccttgtcgt caggccttcg ataccgactc
     5881 cattgagtaa ttccagagca gagtttatga ctttgctctt gtccagtcta gacatcttat
     5941 cgtcatcgtc tttgtaatcc atggtggcgg atcccgcgtc acgacacctg tgttctggcg
     6001 gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg cacttatata cggttctccc
     6061 ccaccctcgg gaaaaaggcg gagccagtac acgacatcac tttcccagtt taccccgcgc
     6121 caccttctct aggcaccggt tcaattgccg acccctcccc ccaacttctc ggggactgtg
     6181 ggcgatgtgc gctctgccca ctgacgggca ccggagccac tcgagtggaa tt
//
