LOCUS       sequence-365622-        7895 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 
            (PhCMV-SEAP-p2A-iRFP670-pA::PmPGK1-IgkSS-Nluc-p2A-mTagBFP2-pA::A3-pA
            ). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7895)
  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 7895)
  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: pTS1042
FEATURES             Location/Qualifiers
     source          1..7895
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     613..632
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(851..955)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1025..1404
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1405..1608
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1559..1579
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1653..1672
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1653..1671
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3290..3346
                     /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             3353..4282
                     /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(4343..4360)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4349..4573
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4621..5120
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4656..4679)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5053..5072
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     5173..5229
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             5251..5763
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             5791..5847
                     /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             5866..6573
                     /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(6695..6711)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6695..6711)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    6867..6888
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7005..7022)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7176..7764)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7256..7275)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7885..7895,1..781,782..850))
                     /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"
ORIGIN
        1 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
       61 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
      121 tgataccgcg gctcccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
      181 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
      241 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
      301 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
      361 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
      421 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
      481 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
      541 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
      601 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
      661 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
      721 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
      781 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
      841 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
      901 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
      961 ttccccgaaa agtgccacct gacgtctaag aaaccaagat ctccacgcgt ggtaccctcg
     1021 agttgacatt gattattgag tagttattaa tagtaatcaa ttacggggtc attagttcat
     1081 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     1141 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     1201 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     1261 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     1321 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     1381 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     1441 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     1501 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     1561 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
     1621 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
     1681 gctgttcgaa gcggaattca ccatgactag tctgctgctg ctgctgctgg gcctgagact
     1741 gcagctgagc ctgggcatca tccccgtgga ggaggagaac cccgacttct ggaacagaga
     1801 ggccgccgag gccctgggcg ccgccaagaa gctgcagccc gcccagaccg ccgccaagaa
     1861 cctgatcatc ttcctgggcg acggcatggg cgtgagcacc gtgaccgccg ccagaatcct
     1921 gaagggccag aagaaggaca agctgggccc cgagatcccc ctggccatgg acagattccc
     1981 ctacgtggcc ctgagcaaga cctacaacgt ggacaagcac gtgcccgaca gcggcgccac
     2041 cgccaccgcc tacctgtgcg gcgtgaaggg caacttccag accatcggcc tgagcgccgc
     2101 cgccagattc aaccagtgca acaccaccag aggcaacgag gtgatcagcg tgatgaacag
     2161 agccaagaag gccggcaaga gcgtgggcgt ggtgaccacc accagagtgc agcacgccag
     2221 ccccgccggc acctacgccc acaccgtgaa cagaaactgg tacagcgacg ccgacgtgcc
     2281 cgccagcgcc agacaggagg gctgccagga catcgccacc cagctgatca gcaacatgga
     2341 catcgacgtg atcctgggcg gcggcagaaa gtacatgttc agaatgggca cccccgaccc
     2401 cgagtacccc gacgactaca gccagggcgg caccagactg gacggcaaga acctggtgca
     2461 ggagtggctg gccaagagac agggcgccag atacgtgtgg aacagaaccg agctgatgca
     2521 ggccagcctg gaccccagcg tgacccacct gatgggcctg ttcgagcccg gcgacatgaa
     2581 gtacgagatc cacagagaca gcaccctgga ccccagcctg atggagatga ccgaggccgc
     2641 cctgagactg ctgagcagaa accccagagg cttcttcctg ttcgtggagg gcggcagaat
     2701 cgaccacggc caccacgaga gcagagccta cagagccctg accgagacca tcatgttcga
     2761 cgacgccatc gagagagccg gccagctgac cagcgaggag gacaccctga gcctggtgac
     2821 cgccgaccac agccacgtgt tcagcttcgg cggctacccc ctgagaggca gcagcatctt
     2881 cggcctggcc cccggcaagg ccagagacag aaaggcctac accgtgctgc tgtacggcaa
     2941 cggccccggc tacgtgctga aggacggcgc cagacccgac gtgaccgaga gcgagagcgg
     3001 cagccccgag tacagacagc agagcgccgt gcccctggac gaggagaccc acgccggcga
     3061 ggacgtggcc gtgttcgcca gaggccccca ggcccacctg gtgcacggcg tgcaggagca
     3121 gaccttcatc gcccacgtga tggccttcgc cgcctgcctg gagccctaca ccgcctgcga
     3181 cctggccccc cccgccggca ccaccgacgc cgcccacccc ggctacagca gagtgggcgc
     3241 cgccggcaga ttcgagcaga ccgctagtgg tggttctggt ggaagcggag ctactaactt
     3301 cagcctgctg aagcaggctg gtgacgtgga ggagaaccct ggacctgcta gtgcgcgtaa
     3361 ggtcgatctc acctcctgcg atcgcgagcc gatccacatc cccggcagca ttcagccgtg
     3421 cggctgcctg cttgcctgcg acgcgcaggc ggtgcggatc acgcgcatta cggaaaatgc
     3481 cggcgcgttc tttggacgcg aaactccgcg ggtcggtgag ctactcgccg attacttcgg
     3541 cgagaccgaa gcccatgcgc tgcgcaacgc actggcgcag tcctccgatc caaagcgacc
     3601 ggcgctgatc ttcggttggc gcgacggcct gaccggccgc accttcgaca tctcactgca
     3661 tcgccatgac ggtacatcga tcatcgagtt cgagcctgcg gcggccgaac aggccgacaa
     3721 tccgctgcgg ctgacgcggc agatcatcgc gcgcaccaaa gaactgaagt cgctcgaaga
     3781 gatggccgca cgggtgccgc gctatctgca ggcgatgctc ggctatcacc gcgtgatgtt
     3841 gtaccgcttc gcggacgacg gctccgggat ggtgatcggc gaggcgaagc gcagcgacct
     3901 cgagagcttt ctcggtcagc actttccggc gtcgctggtc ccgcagcagg cgcggctact
     3961 gtacttgaag aacgcgatcc gcgtggtctc ggattcgcgc ggcatcagca gccggatcgt
     4021 gcccgagcac gacgcctccg gcgccgcgct cgatctgtcg ttcgcgcacc tgcgcagcat
     4081 ctcgccctgc catctcgaat ttctgcggaa catgggcgtc agcgcctcga tgtcgctgtc
     4141 gatcatcatt gacggcacgc tatggggatt gatcatctgt catcattacg agccgcgtgc
     4201 cgtgccgatg gcgcagcgcg tcgcggccga aatgttcgcc gacttcttat cgctgcactt
     4261 caccgccgcc caccaccaac gcgctagcgg atccaccggt gtctagaaag ctttgaggcc
     4321 ggcctgcagg cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     4381 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     4441 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     4501 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     4561 gtgggctcta tggtagctta ggaggcggaa agaaccagcg cgtggtaccc tcgagctacc
     4621 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
     4681 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
     4741 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
     4801 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
     4861 gtctcagtag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
     4921 ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg
     4981 gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct
     5041 ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt
     5101 cctcatctcc gggcctttcg acctccagcc cgagctcggt ataggttcga agcggaattc
     5161 accatgacta gtgagacaga cacactcctg ctatgggtac tgctgctctg ggttccaggt
     5221 tccactggtg acgctagtgg tggttctggt atggtcttca cactcgaaga tttcgttggg
     5281 gactggcgac agacagccgg ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc
     5341 agtttgtttc agaatctcgg ggtgtccgta actccgatcc aaaggattgt cctgagcggt
     5401 gaaaatgggc tgaagatcga catccatgtc atcatcccgt atgaaggtct gagcggcgac
     5461 caaatgggcc agatcgaaaa aatttttaag gtggtgtacc ctgtggatga tcatcacttt
     5521 aaggtgatcc tgcactatgg cacactggta atcgacgggg ttacgccgaa catgatcgac
     5581 tatttcggac ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca
     5641 gggaccctgt ggaacggcaa caaaattatc gacgagcgcc tgatcaaccc cgacggctcc
     5701 ctgctgttcc gagtaaccat caacggagtg accggctggc ggctgtgcga acgcattctg
     5761 gcggctagtg gtggttctgg tggaagcgga gctactaact tcagcctgct gaagcaggct
     5821 ggtgacgtgg aggagaaccc tggacctgct agtggtggtt ctggtgtgtc taagggcgaa
     5881 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
     5941 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     6001 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
     6061 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
     6121 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     6181 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     6241 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     6301 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
     6361 aagctcgtgg gcgggagcca tctgatcgca aacgccaaga ccacatatag atccaagaaa
     6421 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
     6481 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
     6541 gacctcccta gcaaactggg gcacaaactt aatgctagcg gatccaccgg tgtctagaaa
     6601 gctatacagg tgcaccgctt tataataaaa ctgatacaga gaacgttatc atgtgtgttg
     6661 gttttttttg tgtgtgcggt ttaagggagc ctcggtcata gctgtttcct gtgtaaaacg
     6721 acggccagcg cgggagacgg tcagttcgtc tccagctcag aaattatgag agcgtttaat
     6781 aaagtattga ctatggttga caaaacgtgt gttggagttt tttgtgtgct ttgtcatcaa
     6841 ctgagccagt aggtcgaggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     6901 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     6961 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     7021 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     7081 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     7141 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     7201 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     7261 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     7321 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     7381 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     7441 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     7501 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     7561 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     7621 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     7681 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     7741 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gattatcaaa
     7801 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     7861 atatgagtaa acttggtctg acagttacca atgct
//
