LOCUS       sequence-366195-        5172 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 mRuby2 marker and PuroR 
            resistance gene 
            (5'ITR-A1-pA::A2-pA::PRPBSA-mRuby2-p2A-PuroR-pA-3'ITR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5172)
  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 5172)
  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: pTS2354_Tier3(PB)-Ruby_PuroR
FEATURES             Location/Qualifiers
     source          1..5172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             257..313
                     /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             332..925
                     /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     825..845
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    1065..1086
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   1416..1450
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     rep_origin      complement(1522..2110)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1602..1621)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2231..3022,3023..3091))
                     /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     2854..2873
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3092..3196)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   3256..3318
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3612..3629)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3618..3842
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3988..4004)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3988..4004)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4694..5172,1..229)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHM
                     ALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVA
                     KFAGLGGGMDELYK"
ORIGIN
        1 cactcatatg gcactgaaag ttgatggtgg tggccatctg tcttgctctt tcgtaacaac
       61 ttacaggtca aaaaagaccg tcgggaacat caagatgccc ggtatccatg ccgttgatca
      121 ccgcctggaa aggttagagg aaagtgacaa tgaaatgttc gtagtacaac gcgaacacgc
      181 agttgccaag ttcgccgggc ttggtggtgg gatggacgag ctgtacaagg ctagtggtgg
      241 ttctggtgga agcggagcta ctaacttcag cctgctgaag caggctggtg acgtggagga
      301 gaaccctgga cctgctagtg gtggttctgg taccgagtac aagcccacgg tgcgcctcgc
      361 cacccgcgac gacgtccccc gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
      421 cgccacgcgc cacaccgtcg acccggaccg ccacatcgag cgggtcaccg agctgcaaga
      481 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
      541 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
      601 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
      661 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtgtc
      721 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
      781 cgagcgcgcc ggggtgcccg ccttcctgga gacatccgcg ccccgcaacc tccccttcta
      841 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
      901 gtgcatgacc cgcaagcccg gtgccgctag cggatccacc ggtgtctaga aagctcagaa
      961 attatgagag cgtttaataa agtattgact atggttgaca aaacgtgtgt tggagttttt
     1021 tgtgtgcttt gtcatcaact gagccagtag gtcgagggtg cctaatgagt gagctaactc
     1081 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1141 cgatatctat aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat
     1201 aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc
     1261 attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca
     1321 cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc
     1381 gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat
     1441 ctttctaggg ttaaccatgg acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     1501 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     1561 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     1621 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     1681 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     1741 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     1801 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     1861 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     1921 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     1981 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     2041 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     2101 aggatctcaa gaagatcctt tgatcttttc tacggggatt atcaaaaagg atcttcacct
     2161 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     2221 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     2281 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     2341 catctggccc cagtgctgca atgataccgc ggctcccacg ctcaccggct ccagatttat
     2401 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     2461 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     2521 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     2581 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     2641 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     2701 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     2761 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     2821 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     2881 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     2941 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3001 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3061 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     3121 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3181 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccaaga
     3241 tcagcggccg cttaacccta gaaagataat catattgtga cgtacgttaa agataatcat
     3301 gcgtaaaatt gacgcatgtg ttttatcggt ctgtatatcg aggtttattt attaatttga
     3361 atagatatta agttttatta tatttacact tacatactaa taataaattc aacaaacaat
     3421 ttatttatgt ttatttattt attaaaaaaa aacaaaaact caaaatttct tctataaagt
     3481 aacaaaacct taagagatct acgcgtggta ccctcgagga gctcttcgaa gcggaattca
     3541 ccatgactag tgctagcgga tccaccggtg tctagaaagc tttgaggccg gcctgcaggc
     3601 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3661 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3721 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3781 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3841 ggtagcttag gaggcggaaa gaaccagcgc gggagaccgt cagttggtct ccagctatac
     3901 aggtgcaccg ctttataata aaactgatac agagaacgtt atcatgtgtg ttggtttttt
     3961 ttgtgtgtgc ggtttaaggg agcctcggtc atagctgttt cctgtgtaaa acgacggcca
     4021 gcgcgtggta ccctcgagcg tgaccctgtc tcacaaaata aagtaagccc ggactgagtg
     4081 cggaaaggcg ggcctggcgg gtctggactc cccatgcggg ccaccagagg ccctgcagcc
     4141 ttcagtcgct tgaaggggta atggcgcttc cactcacaaa catggcggac agagcgtgtg
     4201 aacgagatga acagcccctc aaaaatatgg ccgccgaggc tggacggccg tgccccagca
     4261 gcaccgcctc cgcgccccac gtgatctctc gccgggcaca gcgctgaccg cggaggtcca
     4321 accggaagaa tgtccggatt ggacattcgg aagagggccc gccttccctg gggaatctct
     4381 gcgcacgcgc agaacgcttc gaccaatgaa aacacaggaa gccgtccgcg caaccgcgtt
     4441 gcgtcacttc tgccgcccct gtttcaaggt atatagccgt agacggaact tcgcctttct
     4501 ctcggcctta gcgccatttt tttgggtgag tgttttttgg ttcctgcgtt gggattccgt
     4561 gtacaatcca tagacatctg acctcggcac ttagcatcat cacagcaaac taactgtagc
     4621 ctttctctct ttccctgtag aaacctctgc acctagggag ctcgaattca ccatgactag
     4681 tggtggttct ggtgtgtcta agggcgaaga gctgatcaag gaaaatatgc gtatgaaggt
     4741 ggtcatggaa ggttcggtca acggccacca attcaaatgc acaggtgaag gagaaggcaa
     4801 tccgtacatg ggaactcaaa ccatgaggat caaagtcatc gagggaggac ccctgccatt
     4861 tgcctttgac attcttgcca cgtcgttcat gtatggcagc cgtactttta tcaagtaccc
     4921 gaaaggcatt cctgatttct ttaaacagtc ctttcctgag ggttttactt gggaaagagt
     4981 tacgagatac gaagatggtg gagtcgtcac cgtcatgcag gacaccagcc ttgaggatgg
     5041 ctgtctcgtt taccacgtcc aagtcagagg ggtaaacttt ccctccaatg gtcccgtgat
     5101 gcagaagaag accaagggtt gggagcctaa tacagagatg atgtatccag cagatggtgg
     5161 tctgagggga ta
//
