LOCUS       sequence-366201-        5185 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-3 vector for stable stable integration of up to three cassettes
            via sleeping beauty 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 5185)
  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 5185)
  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: pTS2339_Tier3(SB)-Ruby_Puro
FEATURES             Location/Qualifiers
     source          1..5185
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(15..806,807..875))
                     /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     638..657
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(876..980)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1443..1460)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1449..1673
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1819..1835)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1819..1835)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2525..3232
                     /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"
     CDS             3260..3316
                     /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             3335..3928
                     /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     3828..3848
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    4068..4089
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4491..5079)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4571..4590)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
       61 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
      121 ttaccatctg gccccagtgc tgcaatgata ccgcggctcc cacgctcacc ggctccagat
      181 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
      241 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
      301 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
      361 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
      421 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
      481 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
      541 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
      601 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
      661 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
      721 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
      781 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
      841 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
      901 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
      961 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     1021 aagatcagcg gccgctacag ttgaagtcgg aagtttacat acagttaagt tggagtcatt
     1081 aaaactcgtt tttcaactac tccacaaatt tcttgttaac aaacaatagt tttggcaagt
     1141 cagttaggac atctactttg tgcatgacac aagtcatttt tccaacaatt gtttacagac
     1201 agattatttc acttataatt cactgtatca caattccagt gggtcagaag tttacataca
     1261 ctaagttcga ctcctctgca gaatgcggcg atgtttcggt aaggggtccg ctatctagtc
     1321 ttaagagatc tacgcgtggt accctcgagg agctcttcga agcggaattc accatgacta
     1381 gtgctagcgg atccaccggt gtctagaaag ctttgaggcc ggcctgcagg cccgctgatc
     1441 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     1501 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     1561 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     1621 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggtagctta
     1681 ggaggcggaa agaaccagcg cgggagaccg tcagttggtc tccagctata caggtgcacc
     1741 gctttataat aaaactgata cagagaacgt tatcatgtgt gttggttttt tttgtgtgtg
     1801 cggtttaagg gagcctcggt catagctgtt tcctgtgtaa aacgacggcc agcgcgtggt
     1861 accctcgagc gtgaccctgt ctcacaaaat aaagtaagcc cggactgagt gcggaaaggc
     1921 gggcctggcg ggtctggact ccccatgcgg gccaccagag gccctgcagc cttcagtcgc
     1981 ttgaaggggt aatggcgctt ccactcacaa acatggcgga cagagcgtgt gaacgagatg
     2041 aacagcccct caaaaatatg gccgccgagg ctggacggcc gtgccccagc agcaccgcct
     2101 ccgcgcccca cgtgatctct cgccgggcac agcgctgacc gcggaggtcc aaccggaaga
     2161 atgtccggat tggacattcg gaagagggcc cgccttccct ggggaatctc tgcgcacgcg
     2221 cagaacgctt cgaccaatga aaacacagga agccgtccgc gcaaccgcgt tgcgtcactt
     2281 ctgccgcccc tgtttcaagg tatatagccg tagacggaac ttcgcctttc tctcggcctt
     2341 agcgccattt ttttgggtga gtgttttttg gttcctgcgt tgggattccg tgtacaatcc
     2401 atagacatct gacctcggca cttagcatca tcacagcaaa ctaactgtag cctttctctc
     2461 tttccctgta gaaacctctg cacctaggga gctcgaattc accatgacta gtggtggttc
     2521 tggtgtgtct aagggcgaag agctgatcaa ggaaaatatg cgtatgaagg tggtcatgga
     2581 aggttcggtc aacggccacc aattcaaatg cacaggtgaa ggagaaggca atccgtacat
     2641 gggaactcaa accatgagga tcaaagtcat cgagggagga cccctgccat ttgcctttga
     2701 cattcttgcc acgtcgttca tgtatggcag ccgtactttt atcaagtacc cgaaaggcat
     2761 tcctgatttc tttaaacagt cctttcctga gggttttact tgggaaagag ttacgagata
     2821 cgaagatggt ggagtcgtca ccgtcatgca ggacaccagc cttgaggatg gctgtctcgt
     2881 ttaccacgtc caagtcagag gggtaaactt tccctccaat ggtcccgtga tgcagaagaa
     2941 gaccaagggt tgggagccta atacagagat gatgtatcca gcagatggtg gtctgagggg
     3001 atacactcat atggcactga aagttgatgg tggtggccat ctgtcttgct ctttcgtaac
     3061 aacttacagg tcaaaaaaga ccgtcgggaa catcaagatg cccggtatcc atgccgttga
     3121 tcaccgcctg gaaaggttag aggaaagtga caatgaaatg ttcgtagtac aacgcgaaca
     3181 cgcagttgcc aagttcgccg ggcttggtgg tgggatggac gagctgtaca aggctagtgg
     3241 tggttctggt ggaagcggag ctactaactt cagcctgctg aagcaggctg gtgacgtgga
     3301 ggagaaccct ggacctgcta gtggtggttc tggtaccgag tacaagccca cggtgcgcct
     3361 cgccacccgc gacgacgtcc cccgggccgt acgcaccctc gccgccgcgt tcgccgacta
     3421 ccccgccacg cgccacaccg tcgacccgga ccgccacatc gagcgggtca ccgagctgca
     3481 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     3541 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     3601 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     3661 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
     3721 gtcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     3781 ggccgagcgc gccggggtgc ccgccttcct ggagacatcc gcgccccgca acctcccctt
     3841 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     3901 ctggtgcatg acccgcaagc ccggtgccgc tagcggatcc accggtgtct agaaagctca
     3961 gaaattatga gagcgtttaa taaagtattg actatggttg acaaaacgtg tgttggagtt
     4021 ttttgtgtgc tttgtcatca actgagccag taggtcgagg gtgcctaatg agtgagctaa
     4081 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4141 ctgctagcta ctcgggaccc cttaccgaaa catcgccgca ttctgcagag gagtcgagtg
     4201 tatgtaaact tctgacccac tgggaatgtg atgaaagaaa taaaagctga aatgaatcat
     4261 tctctctact attattctga tatttcacat tcttaaaata aagtggtgat cctaactgac
     4321 ctaagacagg gaatttttac taggattaaa tgtcaggaat tgtgaaaaag tgagtttaaa
     4381 tgtatttggc taaggtgtat gtaaacttcc gacttcaact gtaccatgga catgtgagca
     4441 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4501 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4561 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4621 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4681 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4741 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4801 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4861 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4921 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4981 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5041 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5101 acggggatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5161 atcaatctaa agtatatatg agtaa
//
