LOCUS       sequence-372399-        8339 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' CoTC 
            insulator sequence 
            (PhCMV-SEAP-p2A-iRFP670-pA::CoTC-PmPGK1-IgkSS-Nluc-p2A-mTagBFP2-pA::
            A3-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8339)
  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 8339)
  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: pTS2321
FEATURES             Location/Qualifiers
     source          1..8339
                     /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"
     promoter        5076..5575
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5111..5134)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5508..5527
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     5628..5684
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             5706..6218
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             6246..6302
                     /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             6321..7028
                     /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(7150..7166)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7150..7166)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    7322..7343
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7460..7477)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7631..8219)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7711..7730)
                     /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 gcgtatgtcc
     4621 cttatggtgc ttctggctct gcagttatta gcatagtgtt accatcaacc accttaactt
     4681 catttttctt attcaatacc taggtaggta gatgctagat tctggaaata aaatatgagt
     4741 ctcaagtggt ccttgtcctc tctcccagtc aaattctgaa tctagttggc aagattctga
     4801 aatcaaggca tataatcagt aataagtgat gatagaaggg tatatagaag aattttatta
     4861 tatgagaggg tgaaaccctc aaaatgaaat gaaatcagac ccttgtctta caccataaac
     4921 aaaaataaat ttgaatgggt taaagaatta aactaagacc taaaaccata aaaattttta
     4981 aagaaatcaa aagaagaaaa ttctaatatt cacgttgcag ccgttttttg aatttgatat
     5041 gagaagcaaa ggcaacaagg taccctcgag ctaccgggta ggggaggcgc ttttcccaag
     5101 gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct
     5161 ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg
     5221 ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct
     5281 cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc agtagtctcg tgcagatgga
     5341 cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg
     5401 ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg
     5461 cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct
     5521 tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcgacctc
     5581 cagcccgagc tcggtatagg ttcgaagcgg aattcaccat gactagtgag acagacacac
     5641 tcctgctatg ggtactgctg ctctgggttc caggttccac tggtgacgct agtggtggtt
     5701 ctggtatggt cttcacactc gaagatttcg ttggggactg gcgacagaca gccggctaca
     5761 acctggacca agtccttgaa cagggaggtg tgtccagttt gtttcagaat ctcggggtgt
     5821 ccgtaactcc gatccaaagg attgtcctga gcggtgaaaa tgggctgaag atcgacatcc
     5881 atgtcatcat cccgtatgaa ggtctgagcg gcgaccaaat gggccagatc gaaaaaattt
     5941 ttaaggtggt gtaccctgtg gatgatcatc actttaaggt gatcctgcac tatggcacac
     6001 tggtaatcga cggggttacg ccgaacatga tcgactattt cggacggccg tatgaaggca
     6061 tcgccgtgtt cgacggcaaa aagatcactg taacagggac cctgtggaac ggcaacaaaa
     6121 ttatcgacga gcgcctgatc aaccccgacg gctccctgct gttccgagta accatcaacg
     6181 gagtgaccgg ctggcggctg tgcgaacgca ttctggcggc tagtggtggt tctggtggaa
     6241 gcggagctac taacttcagc ctgctgaagc aggctggtga cgtggaggag aaccctggac
     6301 ctgctagtgg tggttctggt gtgtctaagg gcgaagagct gattaaggag aacatgcaca
     6361 tgaagctgta catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg
     6421 aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc
     6481 tccccttcgc cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca
     6541 accacaccca gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg
     6601 agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc
     6661 aggacggctg cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc
     6721 ctgtgatgca gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg
     6781 acggcggcct ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga
     6841 tcgcaaacgc caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg
     6901 gcgtctacta tgtggactac agactggaaa gaatcaagga ggccaacaac gagacctacg
     6961 tcgagcagca cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca
     7021 aacttaatgc tagcggatcc accggtgtct agaaagctat acaggtgcac cgctttataa
     7081 taaaactgat acagagaacg ttatcatgtg tgttggtttt ttttgtgtgt gcggtttaag
     7141 ggagcctcgg tcatagctgt ttcctgtgta aaacgacggc cagcgcggga gacggtcagt
     7201 tcgtctccag ctcagaaatt atgagagcgt ttaataaagt attgactatg gttgacaaaa
     7261 cgtgtgttgg agttttttgt gtgctttgtc atcaactgag ccagtaggtc gagggtgcct
     7321 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     7381 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     7441 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     7501 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     7561 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     7621 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     7681 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     7741 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     7801 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     7861 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     7921 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     7981 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     8041 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     8101 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     8161 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     8221 aagatccttt gatcttttct acggggatta tcaaaaagga tcttcaccta gatcctttta
     8281 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacag
//
