LOCUS       sequence-364985-        5395 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 iRFP670 marker and PuroR
            resistance gene 
            (5'ITR-A1-pA::A2-pA::PRPBSA-iRFP670-p2A-PuroR-pA-3'ITR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5395)
  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 5395)
  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: pTS2340_Tier3(SB)-iRFP_Puro
FEATURES             Location/Qualifiers
     source          1..5395
                     /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
     primer_bind     complement(1441..1458)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1447..1671
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1817..1833)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1817..1833)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2511..3440
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="ARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAGA
                     FFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLHR
                     HDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVML
                     YRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSRI
                     VPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYEP
                     RAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     CDS             3468..3524
                     /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             3543..4136
                     /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     4036..4056
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    4276..4297
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4699..5287)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4779..4798)
                     /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 cgctacagtt gaagtcggaa gtttacatac agttaagttg gagtcattaa
     1081 aactcgtttt tcaactactc cacaaatttc ttgttaacaa acaatagttt tggcaagtca
     1141 gttaggacat ctactttgtg catgacacaa gtcatttttc caacaattgt ttacagacag
     1201 attatttcac ttataattca ctgtatcaca attccagtgg gtcagaagtt tacatacact
     1261 aagttcgact cctctgcaga atgcggcgat gtttcggtaa ggggtccgct atctagtctt
     1321 aagagatcta cgcgtggtac cctcgaggag ctcttcgaag cggaattcac catgactagt
     1381 gctagcggat ccaccggtgt ctagaaagct ttgaggccgg cctgcaggcc cgctgatcag
     1441 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     1501 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     1561 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     1621 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gtagcttagg
     1681 aggcggaaag aaccagcgcg ggagaccgtc agttggtctc cagctataca ggtgcaccgc
     1741 tttataataa aactgataca gagaacgtta tcatgtgtgt tggttttttt tgtgtgtgcg
     1801 gtttaaggga gcctcggtca tagctgtttc ctgtgtaaaa cgacggccag cgcgtggtac
     1861 cctcgagcgt gaccctgtct cacaaaataa agtaagcccg gactgagtgc ggaaaggcgg
     1921 gcctggcggg tctggactcc ccatgcgggc caccagaggc cctgcagcct tcagtcgctt
     1981 gaaggggtaa tggcgcttcc actcacaaac atggcggaca gagcgtgtga acgagatgaa
     2041 cagcccctca aaaatatggc cgccgaggct ggacggccgt gccccagcag caccgcctcc
     2101 gcgccccacg tgatctctcg ccgggcacag cgctgaccgc ggaggtccaa ccggaagaat
     2161 gtccggattg gacattcgga agagggcccg ccttccctgg ggaatctctg cgcacgcgca
     2221 gaacgcttcg accaatgaaa acacaggaag ccgtccgcgc aaccgcgttg cgtcacttct
     2281 gccgcccctg tttcaaggta tatagccgta gacggaactt cgcctttctc tcggccttag
     2341 cgccattttt ttgggtgagt gttttttggt tcctgcgttg ggattccgtg tacaatccat
     2401 agacatctga cctcggcact tagcatcatc acagcaaact aactgtagcc tttctctctt
     2461 tccctgtaga aacctctgca cctagggagc tcgaattcac catgactagt gcgcgtaagg
     2521 tcgatctcac ctcctgcgat cgcgagccga tccacatccc cggcagcatt cagccgtgcg
     2581 gctgcctgct tgcctgcgac gcgcaggcgg tgcggatcac gcgcattacg gaaaatgccg
     2641 gcgcgttctt tggacgcgaa actccgcggg tcggtgagct actcgccgat tacttcggcg
     2701 agaccgaagc ccatgcgctg cgcaacgcac tggcgcagtc ctccgatcca aagcgaccgg
     2761 cgctgatctt cggttggcgc gacggcctga ccggccgcac cttcgacatc tcactgcatc
     2821 gccatgacgg tacatcgatc atcgagttcg agcctgcggc ggccgaacag gccgacaatc
     2881 cgctgcggct gacgcggcag atcatcgcgc gcaccaaaga actgaagtcg ctcgaagaga
     2941 tggccgcacg ggtgccgcgc tatctgcagg cgatgctcgg ctatcaccgc gtgatgttgt
     3001 accgcttcgc ggacgacggc tccgggatgg tgatcggcga ggcgaagcgc agcgacctcg
     3061 agagctttct cggtcagcac tttccggcgt cgctggtccc gcagcaggcg cggctactgt
     3121 acttgaagaa cgcgatccgc gtggtctcgg attcgcgcgg catcagcagc cggatcgtgc
     3181 ccgagcacga cgcctccggc gccgcgctcg atctgtcgtt cgcgcacctg cgcagcatct
     3241 cgccctgcca tctcgaattt ctgcggaaca tgggcgtcag cgcctcgatg tcgctgtcga
     3301 tcatcattga cggcacgcta tggggattga tcatctgtca tcattacgag ccgcgtgccg
     3361 tgccgatggc gcagcgcgtc gcggccgaaa tgttcgccga cttcttatcg ctgcacttca
     3421 ccgccgccca ccaccaacgc gctagtggtg gttctggtgg aagcggagct actaacttca
     3481 gcctgctgaa gcaggctggt gacgtggagg agaaccctgg acctgctagt ggtggttctg
     3541 gtaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc cgggccgtac
     3601 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gacccggacc
     3661 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     3721 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     3781 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     3841 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     3901 ccgcgtggtt cctggccacc gtcggcgtgt cgcccgacca ccagggcaag ggtctgggca
     3961 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     4021 agacatccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     4081 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgccgcta
     4141 gcggatccac cggtgtctag aaagctcaga aattatgaga gcgtttaata aagtattgac
     4201 tatggttgac aaaacgtgtg ttggagtttt ttgtgtgctt tgtcatcaac tgagccagta
     4261 ggtcgagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4321 tttccagtcg ggaaacctgt cgtgccagct gctagctact cgggacccct taccgaaaca
     4381 tcgccgcatt ctgcagagga gtcgagtgta tgtaaacttc tgacccactg ggaatgtgat
     4441 gaaagaaata aaagctgaaa tgaatcattc tctctactat tattctgata tttcacattc
     4501 ttaaaataaa gtggtgatcc taactgacct aagacaggga atttttacta ggattaaatg
     4561 tcaggaattg tgaaaaagtg agtttaaatg tatttggcta aggtgtatgt aaacttccga
     4621 cttcaactgt accatggaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4681 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4741 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4801 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4861 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4921 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4981 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5041 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5101 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5161 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5221 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5281 atctcaagaa gatcctttga tcttttctac ggggattatc aaaaaggatc ttcacctaga
     5341 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaac
//
