LOCUS       sequence-365626-        8299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-2 co-expression vector encoding PhCMV-driven SEAP coupled to 
            fluorescent reporter iRFP670 and PmPGK1-driven secreted Nluc coupled
            to fluorescent reporter mTagBFP2 that is insulated via a 5' Tactb 
            insulator sequence 
            (PhCMV-SEAP-p2A-iRFP670-pA::Tactb-PmPGK1-IgkSS-Nluc-p2A-mTagBFP2-pA:
            :A3-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8299)
  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 8299)
  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: pTS2322
FEATURES             Location/Qualifiers
     source          1..8299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     sig_peptide     75..131
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             153..665
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             693..749
                     /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             768..1475
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     primer_bind     complement(1597..1613)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1597..1613)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    1769..1790
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1907..1924)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2078..2666)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2158..2177)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2787..3578,3579..3647))
                     /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     3410..3429
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3648..3752)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        3822..4201
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4202..4405
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4356..4376
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4450..4469
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4450..4468
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6087..6143
                     /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             6150..7079
                     /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"
     primer_bind     complement(7140..7157)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7146..7370
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        join(7822..8299,1..22)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(7857..7880)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     8254..8273
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
ORIGIN
        1 ttcctcatct ccgggccttt cgacctccag cccgagctcg gtataggttc gaagcggaat
       61 tcaccatgac tagtgagaca gacacactcc tgctatgggt actgctgctc tgggttccag
      121 gttccactgg tgacgctagt ggtggttctg gtatggtctt cacactcgaa gatttcgttg
      181 gggactggcg acagacagcc ggctacaacc tggaccaagt ccttgaacag ggaggtgtgt
      241 ccagtttgtt tcagaatctc ggggtgtccg taactccgat ccaaaggatt gtcctgagcg
      301 gtgaaaatgg gctgaagatc gacatccatg tcatcatccc gtatgaaggt ctgagcggcg
      361 accaaatggg ccagatcgaa aaaattttta aggtggtgta ccctgtggat gatcatcact
      421 ttaaggtgat cctgcactat ggcacactgg taatcgacgg ggttacgccg aacatgatcg
      481 actatttcgg acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa
      541 cagggaccct gtggaacggc aacaaaatta tcgacgagcg cctgatcaac cccgacggct
      601 ccctgctgtt ccgagtaacc atcaacggag tgaccggctg gcggctgtgc gaacgcattc
      661 tggcggctag tggtggttct ggtggaagcg gagctactaa cttcagcctg ctgaagcagg
      721 ctggtgacgt ggaggagaac cctggacctg ctagtggtgg ttctggtgtg tctaagggcg
      781 aagagctgat taaggagaac atgcacatga agctgtacat ggagggcacc gtggacaacc
      841 atcacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga
      901 gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctactagct
      961 tcctctacgg cagcaagacc ttcatcaacc acacccaggg catccccgac ttcttcaagc
     1021 agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc
     1081 tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca
     1141 gaggggtgaa cttcacatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg
     1201 ccttcaccga gacgctgtac cccgctgacg gcggcctgga aggcagaaac gacatggccc
     1261 tgaagctcgt gggcgggagc catctgatcg caaacgccaa gaccacatat agatccaaga
     1321 aacccgctaa gaacctcaag atgcctggcg tctactatgt ggactacaga ctggaaagaa
     1381 tcaaggaggc caacaacgag acctacgtcg agcagcacga ggtggcagtg gccagatact
     1441 gcgacctccc tagcaaactg gggcacaaac ttaatgctag cggatccacc ggtgtctaga
     1501 aagctataca ggtgcaccgc tttataataa aactgataca gagaacgtta tcatgtgtgt
     1561 tggttttttt tgtgtgtgcg gtttaaggga gcctcggtca tagctgtttc ctgtgtaaaa
     1621 cgacggccag cgcgggagac ggtcagttcg tctccagctc agaaattatg agagcgttta
     1681 ataaagtatt gactatggtt gacaaaacgt gtgttggagt tttttgtgtg ctttgtcatc
     1741 aactgagcca gtaggtcgag ggtgcctaat gagtgagcta actcacatta attgcgttgc
     1801 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     1861 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     1921 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     1981 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2041 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2101 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2161 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2221 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2281 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2341 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2401 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2461 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     2521 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2581 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2641 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggattatca
     2701 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2761 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2821 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2881 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcggct cccacgctca
     2941 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3001 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3061 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3121 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3181 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3241 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3301 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3361 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3421 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3481 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3541 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3601 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3661 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3721 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     3781 gtctaagaaa ccaagatctc cacgcgtggt accctcgagt tgacattgat tattgagtag
     3841 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     3901 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     3961 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     4021 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     4081 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     4141 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     4201 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     4261 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     4321 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     4381 gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg cttactggct
     4441 tatcgaaatt aatacgactc actataggga gacccaagct gttcgaagcg gaattcacca
     4501 tgactagtct gctgctgctg ctgctgggcc tgagactgca gctgagcctg ggcatcatcc
     4561 ccgtggagga ggagaacccc gacttctgga acagagaggc cgccgaggcc ctgggcgccg
     4621 ccaagaagct gcagcccgcc cagaccgccg ccaagaacct gatcatcttc ctgggcgacg
     4681 gcatgggcgt gagcaccgtg accgccgcca gaatcctgaa gggccagaag aaggacaagc
     4741 tgggccccga gatccccctg gccatggaca gattccccta cgtggccctg agcaagacct
     4801 acaacgtgga caagcacgtg cccgacagcg gcgccaccgc caccgcctac ctgtgcggcg
     4861 tgaagggcaa cttccagacc atcggcctga gcgccgccgc cagattcaac cagtgcaaca
     4921 ccaccagagg caacgaggtg atcagcgtga tgaacagagc caagaaggcc ggcaagagcg
     4981 tgggcgtggt gaccaccacc agagtgcagc acgccagccc cgccggcacc tacgcccaca
     5041 ccgtgaacag aaactggtac agcgacgccg acgtgcccgc cagcgccaga caggagggct
     5101 gccaggacat cgccacccag ctgatcagca acatggacat cgacgtgatc ctgggcggcg
     5161 gcagaaagta catgttcaga atgggcaccc ccgaccccga gtaccccgac gactacagcc
     5221 agggcggcac cagactggac ggcaagaacc tggtgcagga gtggctggcc aagagacagg
     5281 gcgccagata cgtgtggaac agaaccgagc tgatgcaggc cagcctggac cccagcgtga
     5341 cccacctgat gggcctgttc gagcccggcg acatgaagta cgagatccac agagacagca
     5401 ccctggaccc cagcctgatg gagatgaccg aggccgccct gagactgctg agcagaaacc
     5461 ccagaggctt cttcctgttc gtggagggcg gcagaatcga ccacggccac cacgagagca
     5521 gagcctacag agccctgacc gagaccatca tgttcgacga cgccatcgag agagccggcc
     5581 agctgaccag cgaggaggac accctgagcc tggtgaccgc cgaccacagc cacgtgttca
     5641 gcttcggcgg ctaccccctg agaggcagca gcatcttcgg cctggccccc ggcaaggcca
     5701 gagacagaaa ggcctacacc gtgctgctgt acggcaacgg ccccggctac gtgctgaagg
     5761 acggcgccag acccgacgtg accgagagcg agagcggcag ccccgagtac agacagcaga
     5821 gcgccgtgcc cctggacgag gagacccacg ccggcgagga cgtggccgtg ttcgccagag
     5881 gcccccaggc ccacctggtg cacggcgtgc aggagcagac cttcatcgcc cacgtgatgg
     5941 ccttcgccgc ctgcctggag ccctacaccg cctgcgacct ggcccccccc gccggcacca
     6001 ccgacgccgc ccaccccggc tacagcagag tgggcgccgc cggcagattc gagcagaccg
     6061 ctagtggtgg ttctggtgga agcggagcta ctaacttcag cctgctgaag caggctggtg
     6121 acgtggagga gaaccctgga cctgctagtg cgcgtaaggt cgatctcacc tcctgcgatc
     6181 gcgagccgat ccacatcccc ggcagcattc agccgtgcgg ctgcctgctt gcctgcgacg
     6241 cgcaggcggt gcggatcacg cgcattacgg aaaatgccgg cgcgttcttt ggacgcgaaa
     6301 ctccgcgggt cggtgagcta ctcgccgatt acttcggcga gaccgaagcc catgcgctgc
     6361 gcaacgcact ggcgcagtcc tccgatccaa agcgaccggc gctgatcttc ggttggcgcg
     6421 acggcctgac cggccgcacc ttcgacatct cactgcatcg ccatgacggt acatcgatca
     6481 tcgagttcga gcctgcggcg gccgaacagg ccgacaatcc gctgcggctg acgcggcaga
     6541 tcatcgcgcg caccaaagaa ctgaagtcgc tcgaagagat ggccgcacgg gtgccgcgct
     6601 atctgcaggc gatgctcggc tatcaccgcg tgatgttgta ccgcttcgcg gacgacggct
     6661 ccgggatggt gatcggcgag gcgaagcgca gcgacctcga gagctttctc ggtcagcact
     6721 ttccggcgtc gctggtcccg cagcaggcgc ggctactgta cttgaagaac gcgatccgcg
     6781 tggtctcgga ttcgcgcggc atcagcagcc ggatcgtgcc cgagcacgac gcctccggcg
     6841 ccgcgctcga tctgtcgttc gcgcacctgc gcagcatctc gccctgccat ctcgaatttc
     6901 tgcggaacat gggcgtcagc gcctcgatgt cgctgtcgat catcattgac ggcacgctat
     6961 ggggattgat catctgtcat cattacgagc cgcgtgccgt gccgatggcg cagcgcgtcg
     7021 cggccgaaat gttcgccgac ttcttatcgc tgcacttcac cgccgcccac caccaacgcg
     7081 ctagcggatc caccggtgtc tagaaagctt tgaggccggc ctgcaggccc gctgatcagc
     7141 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     7201 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     7261 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     7321 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg tagcttagga
     7381 ggcggaaaga accagcgcgt ggtggggcag tgggggccaa cttgtcctta cccagagtgc
     7441 aggtgtgtgg agatccctcc tgccttgaca ttgagcagcc ttagagggtg ggggaggctc
     7501 aggggtcagg tctctgttcc tgcttattgg ggagttcctg gcctggccct tctatgtctc
     7561 cccaggtacc ccagtttttc tgggttcacc cagagtgcag atgcttgagg aggtgggaag
     7621 ggactatttg ggggtgtctg gctcaggtgc catgcctcac tggggctggt tggcacctgc
     7681 atttcctggg agtggggctg tctcagggta gctgggcacg gtgttccctt gagtgggggt
     7741 gtagtgggtg ttcctagctg ccacgccttt gccttcacct atgggatcgt ggctgtcaga
     7801 accaggtacc ctcgagctac cgggtagggg aggcgctttt cccaaggcag tctggagcat
     7861 gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac
     7921 attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac
     7981 cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga
     8041 cgtgacaaat ggaagtagca cgtctcagta gtctcgtgca gatggacagc accgctgagc
     8101 aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct
     8161 gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg
     8221 gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt
     8281 ctgccgcgct gttctcctc
//
