LOCUS       sequence-366196-        5178 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 
            (5'ITR-A1-pA::A2-pA::PRPBSA-YPet-p2A-PuroR-pA-3'ITR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5178)
  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 5178)
  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: pTS2353_Tier3(PB)-YPet_PuroR
FEATURES             Location/Qualifiers
     source          1..5178
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(13..804,805..873))
                     /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     636..655
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(874..978)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   1038..1100
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1394..1411)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1400..1624
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1770..1786)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1770..1786)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2779..2798)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             3217..3273
                     /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             3292..3885
                     /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     3785..3805
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    4025..4046
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   4376..4410
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     rep_origin      complement(4482..5070)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4562..4581)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
       61 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
      121 accatctggc cccagtgctg caatgatacc gcggctccca cgctcaccgg ctccagattt
      181 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
      241 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
      301 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
      361 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
      421 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
      481 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
      541 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
      601 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
      661 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
      721 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
      781 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
      841 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
      901 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
      961 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccaa
     1021 gatcagcggc cgcttaaccc tagaaagata atcatattgt gacgtacgtt aaagataatc
     1081 atgcgtaaaa ttgacgcatg tgttttatcg gtctgtatat cgaggtttat ttattaattt
     1141 gaatagatat taagttttat tatatttaca cttacatact aataataaat tcaacaaaca
     1201 atttatttat gtttatttat ttattaaaaa aaaacaaaaa ctcaaaattt cttctataaa
     1261 gtaacaaaac cttaagagat ctacgcgtgg taccctcgag gagctcttcg aagcggaatt
     1321 caccatgact agtgctagcg gatccaccgg tgtctagaaa gctttgaggc cggcctgcag
     1381 gcccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     1441 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     1501 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     1561 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     1621 atggtagctt aggaggcgga aagaaccagc gcgggagacc gtcagttggt ctccagctat
     1681 acaggtgcac cgctttataa taaaactgat acagagaacg ttatcatgtg tgttggtttt
     1741 ttttgtgtgt gcggtttaag ggagcctcgg tcatagctgt ttcctgtgta aaacgacggc
     1801 cagcgcgtgg taccctcgag cgtgaccctg tctcacaaaa taaagtaagc ccggactgag
     1861 tgcggaaagg cgggcctggc gggtctggac tccccatgcg ggccaccaga ggccctgcag
     1921 ccttcagtcg cttgaagggg taatggcgct tccactcaca aacatggcgg acagagcgtg
     1981 tgaacgagat gaacagcccc tcaaaaatat ggccgccgag gctggacggc cgtgccccag
     2041 cagcaccgcc tccgcgcccc acgtgatctc tcgccgggca cagcgctgac cgcggaggtc
     2101 caaccggaag aatgtccgga ttggacattc ggaagagggc ccgccttccc tggggaatct
     2161 ctgcgcacgc gcagaacgct tcgaccaatg aaaacacagg aagccgtccg cgcaaccgcg
     2221 ttgcgtcact tctgccgccc ctgtttcaag gtatatagcc gtagacggaa cttcgccttt
     2281 ctctcggcct tagcgccatt tttttgggtg agtgtttttt ggttcctgcg ttgggattcc
     2341 gtgtacaatc catagacatc tgacctcggc acttagcatc atcacagcaa actaactgta
     2401 gcctttctct ctttccctgt agaaacctct gcacctaggg agctcgaatt caccatgact
     2461 agtggtggtt ctggtgtgag caaaggcgaa gagctgttca ccggcgtggt gcccatcctg
     2521 gtggagctgg acggcgacgt gaacggccac aagttcagcg tgagcggcga gggcgagggc
     2581 gacgccacct acggcaagct gaccctgaag ctgctgtgca ccaccggcaa gctgcccgtg
     2641 ccctggccca ccctggtgac caccctgggc tacggcgtgc agtgcttcgc ccggtacccc
     2701 gaccacatga agcagcacga cttcttcaag agcgccatgc ccgagggcta cgtgcaggag
     2761 cggaccatct tcttcaagga cgacggcaac tacaagaccc gggccgaggt gaagttcgag
     2821 ggcgacaccc tggtgaaccg gatcgagctg aagggcatcg acttcaagga ggacggcaac
     2881 atcctgggcc acaagctgga gtacaactac aacagccaca acgtgtacat caccgccgac
     2941 aagcagaaga acggcatcaa ggccaacttc aagatccggc acaacatcga ggacggcggc
     3001 gtgcagctgg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     3061 cccgacaacc actacctgag ctaccagagc gccctgttca aggaccccaa cgagaagcgg
     3121 gaccacatgg tgctgctgga gttcctgacc gccgccggca tcaccgaggg catggacgag
     3181 ctgtacaagg ctagtggtgg ttctggtgga agcggagcta ctaacttcag cctgctgaag
     3241 caggctggtg acgtggagga gaaccctgga cctgctagtg gtggttctgg taccgagtac
     3301 aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg caccctcgcc
     3361 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg acccggaccg ccacatcgag
     3421 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     3481 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     3541 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     3601 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     3661 ctggccaccg tcggcgtgtc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     3721 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacatccgcg
     3781 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     3841 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgccgctag cggatccacc
     3901 ggtgtctaga aagctcagaa attatgagag cgtttaataa agtattgact atggttgaca
     3961 aaacgtgtgt tggagttttt tgtgtgcttt gtcatcaact gagccagtag gtcgagggtg
     4021 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4081 gaaacctgtc gtgccagctg cgatatctat aacaagaaaa tatatatata ataagttatc
     4141 acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac
     4201 gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac
     4261 acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa
     4321 atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc
     4381 gtcaatttta cgcagactat ctttctaggg ttaaccatgg acatgtgagc aaaaggccag
     4441 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4501 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4561 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4621 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4681 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4741 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4801 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4861 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4921 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4981 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5041 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggatt
     5101 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5161 aagtatatat gagtaaac
//
