LOCUS       sequence-364560-        4362 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 PuroR resistance gene 
            (5'ITR-A1-pA::A2-pA::PRPBSA-PuroR-pA-3'ITR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4362)
  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 4362)
  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: pTS2356_Tier3(PB)-PuroR
FEATURES             Location/Qualifiers
     source          1..4362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             226..819
                     /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     719..739
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    959..980
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   1310..1344
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     rep_origin      complement(1416..2004)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1496..1515)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2125..2916,2917..2985))
                     /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     2748..2767
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2986..3090)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   3150..3212
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3506..3523)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3512..3736
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3882..3898)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3882..3898)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gtatatagcc gtagacggaa cttcgccttt ctctcggcct tagcgccatt tttttgggtg
       61 agtgtttttt ggttcctgcg ttgggattcc gtgtacaatc catagacatc tgacctcggc
      121 acttagcatc atcacagcaa actaactgta gcctttctct ctttccctgt agaaacctct
      181 gcacctaggg agctcgaatt caccatgact agtggtggtt ctggtaccga gtacaagccc
      241 acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg
      301 ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc
      361 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
      421 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
      481 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
      541 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
      601 accgtcggcg tgtcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
      661 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacatc cgcgccccgc
      721 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
      781 ggaccgcgca cctggtgcat gacccgcaag cccggtgccg ctagcggatc caccggtgtc
      841 tagaaagctc agaaattatg agagcgttta ataaagtatt gactatggtt gacaaaacgt
      901 gtgttggagt tttttgtgtg ctttgtcatc aactgagcca gtaggtcgag ggtgcctaat
      961 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     1021 tgtcgtgcca gctgcgatat ctataacaag aaaatatata tataataagt tatcacgtaa
     1081 gtagaacatg aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa
     1141 gataatcatg cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac
     1201 cgcattgaca agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac
     1261 agcgacggat tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat
     1321 tttacgcaga ctatctttct agggttaacc atggacatgt gagcaaaagg ccagcaaaag
     1381 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     1441 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     1501 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     1561 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     1621 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     1681 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     1741 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     1801 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     1861 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     1921 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     1981 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gattatcaaa
     2041 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     2101 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     2161 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     2221 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcggctcc cacgctcacc
     2281 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     2341 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     2401 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     2461 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     2521 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     2581 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     2641 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     2701 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     2761 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     2821 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     2881 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     2941 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     3001 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     3061 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     3121 ctaagaaacc aagatcagcg gccgcttaac cctagaaaga taatcatatt gtgacgtacg
     3181 ttaaagataa tcatgcgtaa aattgacgca tgtgttttat cggtctgtat atcgaggttt
     3241 atttattaat ttgaatagat attaagtttt attatattta cacttacata ctaataataa
     3301 attcaacaaa caatttattt atgtttattt atttattaaa aaaaaacaaa aactcaaaat
     3361 ttcttctata aagtaacaaa accttaagag atctacgcgt ggtaccctcg aggagctctt
     3421 cgaagcggaa ttcaccatga ctagtgctag cggatccacc ggtgtctaga aagctttgag
     3481 gccggcctgc aggcccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     3541 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     3601 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     3661 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     3721 gcggtgggct ctatggtagc ttaggaggcg gaaagaacca gcgcgggaga ccgtcagttg
     3781 gtctccagct atacaggtgc accgctttat aataaaactg atacagagaa cgttatcatg
     3841 tgtgttggtt ttttttgtgt gtgcggttta agggagcctc ggtcatagct gtttcctgtg
     3901 taaaacgacg gccagcgcgt ggtaccctcg agcgtgaccc tgtctcacaa aataaagtaa
     3961 gcccggactg agtgcggaaa ggcgggcctg gcgggtctgg actccccatg cgggccacca
     4021 gaggccctgc agccttcagt cgcttgaagg ggtaatggcg cttccactca caaacatggc
     4081 ggacagagcg tgtgaacgag atgaacagcc cctcaaaaat atggccgccg aggctggacg
     4141 gccgtgcccc agcagcaccg cctccgcgcc ccacgtgatc tctcgccggg cacagcgctg
     4201 accgcggagg tccaaccgga agaatgtccg gattggacat tcggaagagg gcccgccttc
     4261 cctggggaat ctctgcgcac gcgcagaacg cttcgaccaa tgaaaacaca ggaagccgtc
     4321 cgcgcaaccg cgttgcgtca cttctgccgc ccctgtttca ag
//
