LOCUS       sequence-366219-        4975 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 mRuby marker and BlastR 
            resistance gene 
            (5'ITR-A1-pA::A2-pA::PRPBSA-mRuby2-p2A-BlastR-pA-3'ITR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4975)
  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 4975)
  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: pTS2344_Tier3(SB)-mRuby2_Blast
FEATURES             Location/Qualifiers
     source          1..4975
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(242..259)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    248..472
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(618..634)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(618..634)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1324..2031
                     /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             2059..2115
                     /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"
     protein_bind    2657..2678
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3080..3668)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3160..3179)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3789..4580,4581..4649))
                     /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     4412..4431
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4650..4754)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gattatttca cttataattc actgtatcac aattccagtg ggtcagaagt ttacatacac
       61 taagttcgac tcctctgcag aatgcggcga tgtttcggta aggggtccgc tatctagtct
      121 taagagatct acgcgtggta ccctcgagga gctcttcgaa gcggaattca ccatgactag
      181 tgctagcgga tccaccggtg tctagaaagc tttgaggccg gcctgcaggc ccgctgatca
      241 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
      301 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
      361 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
      421 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggtagcttag
      481 gaggcggaaa gaaccagcgc gggagaccgt cagttggtct ccagctatac aggtgcaccg
      541 ctttataata aaactgatac agagaacgtt atcatgtgtg ttggtttttt ttgtgtgtgc
      601 ggtttaaggg agcctcggtc atagctgttt cctgtgtaaa acgacggcca gcgcgtggta
      661 ccctcgagcg tgaccctgtc tcacaaaata aagtaagccc ggactgagtg cggaaaggcg
      721 ggcctggcgg gtctggactc cccatgcggg ccaccagagg ccctgcagcc ttcagtcgct
      781 tgaaggggta atggcgcttc cactcacaaa catggcggac agagcgtgtg aacgagatga
      841 acagcccctc aaaaatatgg ccgccgaggc tggacggccg tgccccagca gcaccgcctc
      901 cgcgccccac gtgatctctc gccgggcaca gcgctgaccg cggaggtcca accggaagaa
      961 tgtccggatt ggacattcgg aagagggccc gccttccctg gggaatctct gcgcacgcgc
     1021 agaacgcttc gaccaatgaa aacacaggaa gccgtccgcg caaccgcgtt gcgtcacttc
     1081 tgccgcccct gtttcaaggt atatagccgt agacggaact tcgcctttct ctcggcctta
     1141 gcgccatttt tttgggtgag tgttttttgg ttcctgcgtt gggattccgt gtacaatcca
     1201 tagacatctg acctcggcac ttagcatcat cacagcaaac taactgtagc ctttctctct
     1261 ttccctgtag aaacctctgc acctagggag ctcgaattca ccatgactag tggtggttct
     1321 ggtgtgtcta agggcgaaga gctgatcaag gaaaatatgc gtatgaaggt ggtcatggaa
     1381 ggttcggtca acggccacca attcaaatgc acaggtgaag gagaaggcaa tccgtacatg
     1441 ggaactcaaa ccatgaggat caaagtcatc gagggaggac ccctgccatt tgcctttgac
     1501 attcttgcca cgtcgttcat gtatggcagc cgtactttta tcaagtaccc gaaaggcatt
     1561 cctgatttct ttaaacagtc ctttcctgag ggttttactt gggaaagagt tacgagatac
     1621 gaagatggtg gagtcgtcac cgtcatgcag gacaccagcc ttgaggatgg ctgtctcgtt
     1681 taccacgtcc aagtcagagg ggtaaacttt ccctccaatg gtcccgtgat gcagaagaag
     1741 accaagggtt gggagcctaa tacagagatg atgtatccag cagatggtgg tctgagggga
     1801 tacactcata tggcactgaa agttgatggt ggtggccatc tgtcttgctc tttcgtaaca
     1861 acttacaggt caaaaaagac cgtcgggaac atcaagatgc ccggtatcca tgccgttgat
     1921 caccgcctgg aaaggttaga ggaaagtgac aatgaaatgt tcgtagtaca acgcgaacac
     1981 gcagttgcca agttcgccgg gcttggtggt gggatggacg agctgtacaa ggctagtggt
     2041 ggttctggtg gaagcggagc tactaacttc agcctgctga agcaggctgg tgacgtggag
     2101 gagaaccctg gacctgctag tggtggttct ggtttgtctc aagaagaatc caccctcatt
     2161 gaaagagcaa cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc
     2221 gcagctctct ctagcgacgg ccgcatcttc actggtgtca atgtatatca ttttactggg
     2281 ggaccttgtg cagaactcgt ggtgctgggc actgctgctg ctgcggcagc tggcaacctg
     2341 acttgtatcg tcgcgatcgg aaatgagaac aggggcatct tgagcccctg cggacggtgc
     2401 cgacaggtgc ttctcgatct gcatcctggg atcaaagcca tagtgaagga cagtgatgga
     2461 cagccgacgg cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggcgct
     2521 agcggatcca ccggtgtcta gaaagctcag aaattatgag agcgtttaat aaagtattga
     2581 ctatggttga caaaacgtgt gttggagttt tttgtgtgct ttgtcatcaa ctgagccagt
     2641 aggtcgaggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     2701 ctttccagtc gggaaacctg tcgtgccagc tgctagctac tcgggacccc ttaccgaaac
     2761 atcgccgcat tctgcagagg agtcgagtgt atgtaaactt ctgacccact gggaatgtga
     2821 tgaaagaaat aaaagctgaa atgaatcatt ctctctacta ttattctgat atttcacatt
     2881 cttaaaataa agtggtgatc ctaactgacc taagacaggg aatttttact aggattaaat
     2941 gtcaggaatt gtgaaaaagt gagtttaaat gtatttggct aaggtgtatg taaacttccg
     3001 acttcaactg taccatggac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3061 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3121 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3181 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3241 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3301 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3361 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3421 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3481 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3541 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3601 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3661 gatctcaaga agatcctttg atcttttcta cggggattat caaaaaggat cttcacctag
     3721 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3781 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3841 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3901 tctggcccca gtgctgcaat gataccgcgg ctcccacgct caccggctcc agatttatca
     3961 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4021 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4081 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4141 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4201 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     4261 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4321 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4381 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     4441 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4501 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4561 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4621 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4681 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4741 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccaagatc
     4801 agcggccgct acagttgaag tcggaagttt acatacagtt aagttggagt cattaaaact
     4861 cgtttttcaa ctactccaca aatttcttgt taacaaacaa tagttttggc aagtcagtta
     4921 ggacatctac tttgtgcatg acacaagtca tttttccaac aattgtttac agaca
//
