LOCUS       sequence-344441-        5954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one piggyBac transposon vector for dox-inducible expression 
            of Golden 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 5954)
  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 5954)
  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-ERP2
FEATURES             Location/Qualifiers
     source          1..5954
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..376
                     /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    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"
     polyA_signal    623..757
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1033..1067
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1078..1095)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1078..1094)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1087..1109)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1303..1322)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1424..1446
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1485..1503)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1571..1675
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1676..1744,1745..2536)
                     /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(1894..1913)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2707..3295
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3196..3215
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3449..3466
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3672..3693
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3708..3713,3714..3731,3732..3738)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3746..3762
                     /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     3751..3773
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3770..3786
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3770..3786
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   3812..3874
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4050..4131
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4848..4904)
                     /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(4914..5657)
                     /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(5658..5681)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      5681..5690
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5710..5921)
                     /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 ctttgcttat gtaaaccagg gcgcctataa aagagtgctg
      301 attttttgag taaacttcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac
      361 ttcctaccct cgtaaaggta ccgccacccg agaccattaa tgggtctcgt aatgataggc
      421 gtcttcgaat aatggaagac atactagtag accacctccc ctgcgagcta agctggacag
      481 ccaatgacgg gtaagagagt gacatttttc actaacctaa gacaggaggg ccgtcagagc
      541 tactgcctaa tccaaagacg ggtaaaagtg ataaaaatgt atcactccaa cctaagacag
      601 gcgcagcttc cgagggattt gagatccaga catgataaga tacattgatg agtttggaca
      661 aaccaaaact agaatgcagt gaaaaaaatg ccttatttgt gaaatttgtg atgctattgc
      721 cttatttgta accattataa gctgcaataa acaagtttga tatctataac aagaaaatat
      781 atatataata agttatcacg taagtagaac atgaaataac aatataatta tcgtatgagt
      841 taaatcttaa aagtcacgta aaagataatc atgcgtcatt ttgactcacg cggtcgttat
      901 agttcaaaat cagtgacact taccgcattg acaagcacgc ctcacgggag ctccaagcgg
      961 cgactgagat gtcctaaatg cacagcgacg gattcgcgct atttagaaag agagagcaat
     1021 atttcaagaa tgcatgcgtc aattttacgc agactatctt tctagggtta agaattcact
     1081 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     1141 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     1201 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
     1261 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
     1321 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     1381 tctgctcccg gcatccgctt acagacaagc tgtgaccatg tctccgggag ctgcatgtgt
     1441 cagaggtttt caccgtcatc accgaaacac gcgagttcga aagggcctcg tgatacgcct
     1501 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     1561 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1621 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1681 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1741 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1801 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1861 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     1921 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1981 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2041 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2101 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2161 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     2221 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2281 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2341 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gttcacgcgg
     2401 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2461 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2521 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2581 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2641 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2701 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2761 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2821 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     2881 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2941 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3001 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3061 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3121 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3181 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3241 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3301 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3361 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3421 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggatgagcg
     3481 cccaatacgc aaaccgcctc tcaccgcgcg ttggccgatt cattaatgca gctggcacga
     3541 caggtttccc gactggaaag cgggcagtga gcgcaacgca aaccgcctct ccccgcgcgt
     3601 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     3661 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     3721 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     3781 tatgaccatg attacgccaa ggtcgactta accctagaaa gataatcata ttgtgacgta
     3841 cgttaaagat aatcatgcgt aaaattgacg catgtgtttt atcggtctgt atatcgaggt
     3901 ttatttatta atttgaatag atattaagtt ttattatatt tacacttaca tactaataat
     3961 aaattcaaca aacaatttat ttatgtttat ttatttatta aaaaaaaaca aaaactcaaa
     4021 atttcttcta taaagtaaca aaacttttag cagtgaaaaa aatgctttat ttgtgaaatt
     4081 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     4141 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag
     4201 taaaacctct acaaatgtgg tatggctgat tatgatcctc tggagattca tgcgccaggt
     4261 tttctggtca tgcaccacgt tcgtgggccc tcaggaacct ccacgtcagc ggtaactgtg
     4321 aagcccagtc gctcgtaaaa aggaagattc cggggagcac tggtttcgag gaacgccggc
     4381 acgccagccc gttccgcggc ctcaacacca ggaagaacga cagcgcttcc caaaccttta
     4441 ccttggtgat ctgggctgac accgacagtg gcaagaaacc acgcaggctc ctttggtcta
     4501 tgaggagcca gcaagccctc catttgctgt tgtgccgcca acctactccc tgacagctcg
     4561 gccatccgcg gacctatttc cgcgaacact gcgccggctt cgacgctttc cggagtagtc
     4621 cagaccgcaa ctgcggcacc atcgtccgca acccaaactt taccaatgtc cagacccacg
     4681 cgcgtaagaa acagctcttg caactcagtt accctctcta tgtgtctgtc cggatctacg
     4741 gtatggcggg tcgctggata atctgcaaat gctgcggcaa gtgttcggac agcccgtggt
     4801 acatcgtcac gggtagcgag cctaacagtg ggcttatatt ccgtcatggg gccggggttc
     4861 tcctccacgt cgccggcctg cttcagcagg ctgaagttgg tggcgccgct gcccccgggg
     4921 agcatgtcaa ggtcaaaatc gtcaagagcg tcagcaggca gcatatcaag gtcaaagtcg
     4981 tcaagggcat cggctgggag catgtctaag tcaaaatcgt caagggcgtc ggtcggcccg
     5041 ccgctttcgc actttagctg tttctccagg ccacatatga ttagttccag gccgaaaagg
     5101 aaggcaggtt cggctccctg ccggtcgaac agctcaattg cttgtttcag aagtgggggc
     5161 atagaatcgg tggtaggtgt ctctctttcc tcttttgcta cttgatgctc ctgttcctcc
     5221 aatacgcagc ccagtgtaaa gtggcccacg gcggacagag cgtacagtgc gttctccagg
     5281 gagaagcctt gctgacacag gaacgcgagc tgattttcca gggtttcgta ctgtttctct
     5341 gttgggcggg tgccgagatg cactttagcc ccgtcgcgat gtgagaggag agcacagcgg
     5401 tatgacttgg cgttgttccg cagaaagtct tgccatgact cgccttccag ggggcaggag
     5461 tgggtatgat gcctgtccag catctcgatt ggcagggcat cgagcagggc ccgcttgttc
     5521 ttcacgtgcc agtacagggt aggctgctca actcccagct tttgagcgag tttccttgtc
     5581 gtcaggcctt cgataccgac tccattgagt aattccagag cagagtttat gactttgctc
     5641 ttgtccagtc tagacatctt atcgtcatcg tctttgtaat ccatggtggc ggatcccgcg
     5701 tcacgacacc tgtgttctgg cggcaaaccc gttgcgaaaa agaacgttca cggcgactac
     5761 tgcacttata tacggttctc ccccaccctc gggaaaaagg cggagccagt acacgacatc
     5821 actttcccag tttaccccgc gccaccttct ctaggcaccg gttcaattgc cgacccctcc
     5881 ccccaacttc tcggggactg tgggcgatgt gcgctctgcc cactgacggg caccggagcc
     5941 actcgagtgg aatt
//
