LOCUS       sequence-364557-        6494 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-3 vector for stable stable integration of up to three cassettes
            via Piggy Bac transposase including a YPet marker and PuroR 
            resistance gene and a PPGK-driven rtTA transactivator 
            (5'ITR-A1-pA::PPGK-rtTA-pA::PRPBSA-YPet-p2A-PuroR-pA-3'ITR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6494)
  AUTHORS   Haellman V, Strittmatter T, Bertschi A, Stucheli P, Fussenegger M
  TITLE     A versatile plasmid architecture for mammalian synthetic biology 
            (VAMSyB).
  JOURNAL   Metab Eng. 2021 Jul;66:41-50. doi: 10.1016/j.ymben.2021.04.003. Epub
            2021 Apr 18.
  PUBMED    33857582
REFERENCE   2  (bases 1 to 6494)
  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: "Metab 
            Eng."; date: "2021-07"; volume: "66:41-50. doi"; pages: " 
            10.1016/j.ymben.2021.04.003. Epub 2021 Apr 18"
COMMENT     Sequence Label: pTS2363_Tier3(PB)-rtTA-YPet_PuroR
FEATURES             Location/Qualifiers
     source          1..6494
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(77..868,869..937))
                     /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     700..719
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(938..1042)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   1102..1164
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1458..1475)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1464..1688
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1736..2235
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1771..1794)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2168..2187
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2288..3028
                     /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="SRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTD
                     SMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPAD
                     ALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(3150..3166)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3150..3166)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4159..4178)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             4597..4653
                     /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             4672..5265
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     5165..5185
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    5405..5426
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   5756..5790
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     rep_origin      complement(5862..6450)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5942..5961)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
       61 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
      121 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
      181 gcttaccatc tggccccagt gctgcaatga taccgcggct cccacgctca ccggctccag
      241 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
      301 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
      361 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
      421 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
      481 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
      541 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
      601 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
      661 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
      721 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
      781 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
      841 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
      901 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
      961 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     1021 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     1081 ccaagatcag cggccgctta accctagaaa gataatcata ttgtgacgta cgttaaagat
     1141 aatcatgcgt aaaattgacg catgtgtttt atcggtctgt atatcgaggt ttatttatta
     1201 atttgaatag atattaagtt ttattatatt tacacttaca tactaataat aaattcaaca
     1261 aacaatttat ttatgtttat ttatttatta aaaaaaaaca aaaactcaaa atttcttcta
     1321 taaagtaaca aaaccttaag agatctacgc gtggtaccct cgaggagctc ttcgaagcgg
     1381 aattcaccat gactagtgct agcggatcca ccggtgtcta gaaagctttg aggccggcct
     1441 gcaggcccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     1501 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     1561 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     1621 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     1681 ctctatggta gcttaggagg cggaaagaac cagcgcgtgg taccctcgag ctaccgggta
     1741 ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact
     1801 tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa
     1861 ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag
     1921 ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc
     1981 agtagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc
     2041 agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg
     2101 tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga
     2161 ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca
     2221 tctccgggcc tttcgacctc cagcccgagc tcggtatagg gagaccgaat tcaccatgac
     2281 tagtggatca agactggaca agagcaaagt cataaactct gctctggaat tactcaatgg
     2341 agtcggtatc gaaggcctga cgacaaggaa actcgctcaa aagctgggag ttgagcagcc
     2401 taccctgtac tggcacgtga agaacaagcg ggccctgctc gatgccctgc caatcgagat
     2461 gctggacagg catcataccc actcctgccc cctggaaggc gagtcatggc aagactttct
     2521 gcggaacaac gccaagtcat accgctgtgc tctcctctca catcgcgacg gggctaaagt
     2581 gcatctcggc acccgcccaa cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt
     2641 cctgtgtcag caaggcttct ccctggagaa cgcactgtac gctctgtccg ccgtgggcca
     2701 ctttacactg ggctgcgtat tggaggaaca ggagcatcaa gtagcaaaag aggaaagaga
     2761 gacacctacc accgattcta tgcccccact tctgaaacaa gcaattgagc tgttcgaccg
     2821 gcagggagcc gaacctgcct tccttttcgg cctggaacta atcatatgtg gcctggagaa
     2881 acagctaaag tgcgaaagcg gcgggccgac cgacgccctt gacgattttg acttagacat
     2941 gctcccagcc gatgcccttg acgactttga ccttgatatg ctgcctgctg acgctcttga
     3001 cgattttgac cttgacatgc tccccggggc tagcggatcc accggtgtct agaaagctat
     3061 acaggtgcac cgctttataa taaaactgat acagagaacg ttatcatgtg tgttggtttt
     3121 ttttgtgtgt gcggtttaag ggagcctcgg tcatagctgt ttcctgtgta aaacgacggc
     3181 cagcgcgtgg taccctcgag cgtgaccctg tctcacaaaa taaagtaagc ccggactgag
     3241 tgcggaaagg cgggcctggc gggtctggac tccccatgcg ggccaccaga ggccctgcag
     3301 ccttcagtcg cttgaagggg taatggcgct tccactcaca aacatggcgg acagagcgtg
     3361 tgaacgagat gaacagcccc tcaaaaatat ggccgccgag gctggacggc cgtgccccag
     3421 cagcaccgcc tccgcgcccc acgtgatctc tcgccgggca cagcgctgac cgcggaggtc
     3481 caaccggaag aatgtccgga ttggacattc ggaagagggc ccgccttccc tggggaatct
     3541 ctgcgcacgc gcagaacgct tcgaccaatg aaaacacagg aagccgtccg cgcaaccgcg
     3601 ttgcgtcact tctgccgccc ctgtttcaag gtatatagcc gtagacggaa cttcgccttt
     3661 ctctcggcct tagcgccatt tttttgggtg agtgtttttt ggttcctgcg ttgggattcc
     3721 gtgtacaatc catagacatc tgacctcggc acttagcatc atcacagcaa actaactgta
     3781 gcctttctct ctttccctgt agaaacctct gcacctaggg agctcgaatt caccatgact
     3841 agtggtggtt ctggtgtgag caaaggcgaa gagctgttca ccggcgtggt gcccatcctg
     3901 gtggagctgg acggcgacgt gaacggccac aagttcagcg tgagcggcga gggcgagggc
     3961 gacgccacct acggcaagct gaccctgaag ctgctgtgca ccaccggcaa gctgcccgtg
     4021 ccctggccca ccctggtgac caccctgggc tacggcgtgc agtgcttcgc ccggtacccc
     4081 gaccacatga agcagcacga cttcttcaag agcgccatgc ccgagggcta cgtgcaggag
     4141 cggaccatct tcttcaagga cgacggcaac tacaagaccc gggccgaggt gaagttcgag
     4201 ggcgacaccc tggtgaaccg gatcgagctg aagggcatcg acttcaagga ggacggcaac
     4261 atcctgggcc acaagctgga gtacaactac aacagccaca acgtgtacat caccgccgac
     4321 aagcagaaga acggcatcaa ggccaacttc aagatccggc acaacatcga ggacggcggc
     4381 gtgcagctgg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     4441 cccgacaacc actacctgag ctaccagagc gccctgttca aggaccccaa cgagaagcgg
     4501 gaccacatgg tgctgctgga gttcctgacc gccgccggca tcaccgaggg catggacgag
     4561 ctgtacaagg ctagtggtgg ttctggtgga agcggagcta ctaacttcag cctgctgaag
     4621 caggctggtg acgtggagga gaaccctgga cctgctagtg gtggttctgg taccgagtac
     4681 aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg caccctcgcc
     4741 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg acccggaccg ccacatcgag
     4801 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     4861 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     4921 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     4981 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     5041 ctggccaccg tcggcgtgtc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     5101 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacatccgcg
     5161 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     5221 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgccgctag cggatccacc
     5281 ggtgtctaga aagctcagaa attatgagag cgtttaataa agtattgact atggttgaca
     5341 aaacgtgtgt tggagttttt tgtgtgcttt gtcatcaact gagccagtag gtcgagggtg
     5401 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5461 gaaacctgtc gtgccagctg cgatatctat aacaagaaaa tatatatata ataagttatc
     5521 acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac
     5581 gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac
     5641 acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa
     5701 atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc
     5761 gtcaatttta cgcagactat ctttctaggg ttaaccatgg acatgtgagc aaaaggccag
     5821 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5881 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5941 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6001 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6061 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6121 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6181 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6241 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6301 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6361 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6421 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggatt
     6481 atcaaaaagg atct
//
