LOCUS       sequence-366769-        7895 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-2 co-expression vector encoding PhCMV-driven SEAP coupled to 
            fluorescent reporter iRFP670 and PPGK-driven secreted Nluc coupled 
            to fluorescent reporter mTagBFP2 
            (PhCMV-SEAP-p2A-iRFP670-pA::A2-pA::PPGK-IgkSS-Nluc-p2A-mTagBFP2-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: pTS1043
FEATURES             Location/Qualifiers
     source          1..7895
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1..792,793..861))
                     /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     624..643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1036..1415
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1416..1619
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1570..1590
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1664..1683
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1664..1682
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3301..3357
                     /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             3364..4293
                     /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(4354..4371)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4360..4584
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4730..4746)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4730..4746)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4786..5285
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4821..4844)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5218..5237
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     5338..5394
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             5416..5928
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             5956..6012
                     /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             6031..6738
                     /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"
     protein_bind    6878..6899
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7016..7033)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7187..7775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7267..7286)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
       61 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      121 cagtgctgca atgataccgc ggctcccacg ctcaccggct ccagatttat cagcaataaa
      181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
      841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
      901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
      961 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccaaga tctccacgcg
     1021 tggtaccctc gagttgacat tgattattga gtagttatta atagtaatca attacggggt
     1081 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     1141 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     1201 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     1261 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     1321 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     1381 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     1441 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     1501 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     1561 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
     1621 tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg actcactata
     1681 gggagaccca agctgttcga agcggaattc accatgacta gtctgctgct gctgctgctg
     1741 ggcctgagac tgcagctgag cctgggcatc atccccgtgg aggaggagaa ccccgacttc
     1801 tggaacagag aggccgccga ggccctgggc gccgccaaga agctgcagcc cgcccagacc
     1861 gccgccaaga acctgatcat cttcctgggc gacggcatgg gcgtgagcac cgtgaccgcc
     1921 gccagaatcc tgaagggcca gaagaaggac aagctgggcc ccgagatccc cctggccatg
     1981 gacagattcc cctacgtggc cctgagcaag acctacaacg tggacaagca cgtgcccgac
     2041 agcggcgcca ccgccaccgc ctacctgtgc ggcgtgaagg gcaacttcca gaccatcggc
     2101 ctgagcgccg ccgccagatt caaccagtgc aacaccacca gaggcaacga ggtgatcagc
     2161 gtgatgaaca gagccaagaa ggccggcaag agcgtgggcg tggtgaccac caccagagtg
     2221 cagcacgcca gccccgccgg cacctacgcc cacaccgtga acagaaactg gtacagcgac
     2281 gccgacgtgc ccgccagcgc cagacaggag ggctgccagg acatcgccac ccagctgatc
     2341 agcaacatgg acatcgacgt gatcctgggc ggcggcagaa agtacatgtt cagaatgggc
     2401 acccccgacc ccgagtaccc cgacgactac agccagggcg gcaccagact ggacggcaag
     2461 aacctggtgc aggagtggct ggccaagaga cagggcgcca gatacgtgtg gaacagaacc
     2521 gagctgatgc aggccagcct ggaccccagc gtgacccacc tgatgggcct gttcgagccc
     2581 ggcgacatga agtacgagat ccacagagac agcaccctgg accccagcct gatggagatg
     2641 accgaggccg ccctgagact gctgagcaga aaccccagag gcttcttcct gttcgtggag
     2701 ggcggcagaa tcgaccacgg ccaccacgag agcagagcct acagagccct gaccgagacc
     2761 atcatgttcg acgacgccat cgagagagcc ggccagctga ccagcgagga ggacaccctg
     2821 agcctggtga ccgccgacca cagccacgtg ttcagcttcg gcggctaccc cctgagaggc
     2881 agcagcatct tcggcctggc ccccggcaag gccagagaca gaaaggccta caccgtgctg
     2941 ctgtacggca acggccccgg ctacgtgctg aaggacggcg ccagacccga cgtgaccgag
     3001 agcgagagcg gcagccccga gtacagacag cagagcgccg tgcccctgga cgaggagacc
     3061 cacgccggcg aggacgtggc cgtgttcgcc agaggccccc aggcccacct ggtgcacggc
     3121 gtgcaggagc agaccttcat cgcccacgtg atggccttcg ccgcctgcct ggagccctac
     3181 accgcctgcg acctggcccc ccccgccggc accaccgacg ccgcccaccc cggctacagc
     3241 agagtgggcg ccgccggcag attcgagcag accgctagtg gtggttctgg tggaagcgga
     3301 gctactaact tcagcctgct gaagcaggct ggtgacgtgg aggagaaccc tggacctgct
     3361 agtgcgcgta aggtcgatct cacctcctgc gatcgcgagc cgatccacat ccccggcagc
     3421 attcagccgt gcggctgcct gcttgcctgc gacgcgcagg cggtgcggat cacgcgcatt
     3481 acggaaaatg ccggcgcgtt ctttggacgc gaaactccgc gggtcggtga gctactcgcc
     3541 gattacttcg gcgagaccga agcccatgcg ctgcgcaacg cactggcgca gtcctccgat
     3601 ccaaagcgac cggcgctgat cttcggttgg cgcgacggcc tgaccggccg caccttcgac
     3661 atctcactgc atcgccatga cggtacatcg atcatcgagt tcgagcctgc ggcggccgaa
     3721 caggccgaca atccgctgcg gctgacgcgg cagatcatcg cgcgcaccaa agaactgaag
     3781 tcgctcgaag agatggccgc acgggtgccg cgctatctgc aggcgatgct cggctatcac
     3841 cgcgtgatgt tgtaccgctt cgcggacgac ggctccggga tggtgatcgg cgaggcgaag
     3901 cgcagcgacc tcgagagctt tctcggtcag cactttccgg cgtcgctggt cccgcagcag
     3961 gcgcggctac tgtacttgaa gaacgcgatc cgcgtggtct cggattcgcg cggcatcagc
     4021 agccggatcg tgcccgagca cgacgcctcc ggcgccgcgc tcgatctgtc gttcgcgcac
     4081 ctgcgcagca tctcgccctg ccatctcgaa tttctgcgga acatgggcgt cagcgcctcg
     4141 atgtcgctgt cgatcatcat tgacggcacg ctatggggat tgatcatctg tcatcattac
     4201 gagccgcgtg ccgtgccgat ggcgcagcgc gtcgcggccg aaatgttcgc cgacttctta
     4261 tcgctgcact tcaccgccgc ccaccaccaa cgcgctagcg gatccaccgg tgtctagaaa
     4321 gctttgaggc cggcctgcag gcccgctgat cagcctcgac tgtgccttct agttgccagc
     4381 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     4441 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     4501 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     4561 ctggggatgc ggtgggctct atggtagctt aggaggcgga aagaaccagc gcgggagacc
     4621 gtcagttggt ctccagctat acaggtgcac cgctttataa taaaactgat acagagaacg
     4681 ttatcatgtg tgttggtttt ttttgtgtgt gcggtttaag ggagcctcgg tcatagctgt
     4741 ttcctgtgta aaacgacggc cagcgcgtgg taccctcgag ctaccgggta ggggaggcgc
     4801 ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca
     4861 caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt
     4921 tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg
     4981 ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc agtagtctcg
     5041 tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
     5101 agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg
     5161 ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat
     5221 tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc
     5281 tttcgacctc cagcccgagc tcggtatagg ttcgaagcgg aattcaccat gactagtgag
     5341 acagacacac tcctgctatg ggtactgctg ctctgggttc caggttccac tggtgacgct
     5401 agtggtggtt ctggtatggt cttcacactc gaagatttcg ttggggactg gcgacagaca
     5461 gccggctaca acctggacca agtccttgaa cagggaggtg tgtccagttt gtttcagaat
     5521 ctcggggtgt ccgtaactcc gatccaaagg attgtcctga gcggtgaaaa tgggctgaag
     5581 atcgacatcc atgtcatcat cccgtatgaa ggtctgagcg gcgaccaaat gggccagatc
     5641 gaaaaaattt ttaaggtggt gtaccctgtg gatgatcatc actttaaggt gatcctgcac
     5701 tatggcacac tggtaatcga cggggttacg ccgaacatga tcgactattt cggacggccg
     5761 tatgaaggca tcgccgtgtt cgacggcaaa aagatcactg taacagggac cctgtggaac
     5821 ggcaacaaaa ttatcgacga gcgcctgatc aaccccgacg gctccctgct gttccgagta
     5881 accatcaacg gagtgaccgg ctggcggctg tgcgaacgca ttctggcggc tagtggtggt
     5941 tctggtggaa gcggagctac taacttcagc ctgctgaagc aggctggtga cgtggaggag
     6001 aaccctggac ctgctagtgg tggttctggt gtgtctaagg gcgaagagct gattaaggag
     6061 aacatgcaca tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca
     6121 tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag
     6181 ggcggccctc tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag
     6241 accttcatca accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc
     6301 ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac
     6361 accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca
     6421 tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg
     6481 taccccgctg acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg
     6541 agccatctga tcgcaaacgc caagaccaca tatagatcca agaaacccgc taagaacctc
     6601 aagatgcctg gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac
     6661 gagacctacg tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa
     6721 ctggggcaca aacttaatgc tagcggatcc accggtgtct agaaagctca gaaattatga
     6781 gagcgtttaa taaagtattg actatggttg acaaaacgtg tgttggagtt ttttgtgtgc
     6841 tttgtcatca actgagccag taggtcgagg gtgcctaatg agtgagctaa ctcacattaa
     6901 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     6961 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     7021 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     7081 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     7141 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     7201 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     7261 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     7321 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     7381 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     7441 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     7501 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     7561 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     7621 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     7681 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     7741 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     7801 ggattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     7861 atctaaagta tatatgagta aacttggtct gacag
//
