LOCUS       sequence-366778-        8318 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 preluded by a 5' spacer sequence 
            (PhCMV-SEAP-p2A-iRFP670-pA::spacer-PmPGK1-IgkSS-Nluc-p2A-mTagBFP2-pA
            ::A3-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8318)
  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 8318)
  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: pTS2329
FEATURES             Location/Qualifiers
     source          1..8318
                     /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(4724..4745)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4936..4955
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5055..5554
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5090..5113)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5487..5506
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     5607..5663
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             5685..6197
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             6225..6281
                     /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             6300..7007
                     /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(7129..7145)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7129..7145)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    7301..7322
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7439..7456)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7610..8198)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7690..7709)
                     /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 gcgtattcgc
     4621 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     4681 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     4741 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     4801 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     4861 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     4921 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     4981 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggggt
     5041 accctcgagc taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt
     5101 agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac
     5161 atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac
     5221 tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca
     5281 aatggaagta gcacgtctca gtagtctcgt gcagatggac agcaccgctg agcaatggaa
     5341 gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca
     5401 gaggctggga aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg
     5461 cgcccgaagg tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc
     5521 gctgttctcc tcttcctcat ctccgggcct ttcgacctcc agcccgagct cggtataggt
     5581 tcgaagcgga attcaccatg actagtgaga cagacacact cctgctatgg gtactgctgc
     5641 tctgggttcc aggttccact ggtgacgcta gtggtggttc tggtatggtc ttcacactcg
     5701 aagatttcgt tggggactgg cgacagacag ccggctacaa cctggaccaa gtccttgaac
     5761 agggaggtgt gtccagtttg tttcagaatc tcggggtgtc cgtaactccg atccaaagga
     5821 ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag
     5881 gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg taccctgtgg
     5941 atgatcatca ctttaaggtg atcctgcact atggcacact ggtaatcgac ggggttacgc
     6001 cgaacatgat cgactatttc ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa
     6061 agatcactgt aacagggacc ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca
     6121 accccgacgg ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt
     6181 gcgaacgcat tctggcggct agtggtggtt ctggtggaag cggagctact aacttcagcc
     6241 tgctgaagca ggctggtgac gtggaggaga accctggacc tgctagtggt ggttctggtg
     6301 tgtctaaggg cgaagagctg attaaggaga acatgcacat gaagctgtac atggagggca
     6361 ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca
     6421 cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc
     6481 tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag ggcatccccg
     6541 acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag
     6601 acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca
     6661 acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag aagaaaacac
     6721 tcggctggga ggccttcacc gagacgctgt accccgctga cggcggcctg gaaggcagaa
     6781 acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacgcc aagaccacat
     6841 atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat gtggactaca
     6901 gactggaaag aatcaaggag gccaacaacg agacctacgt cgagcagcac gaggtggcag
     6961 tggccagata ctgcgacctc cctagcaaac tggggcacaa acttaatgct agcggatcca
     7021 ccggtgtcta gaaagctata caggtgcacc gctttataat aaaactgata cagagaacgt
     7081 tatcatgtgt gttggttttt tttgtgtgtg cggtttaagg gagcctcggt catagctgtt
     7141 tcctgtgtaa aacgacggcc agcgcgggag acggtcagtt cgtctccagc tcagaaatta
     7201 tgagagcgtt taataaagta ttgactatgg ttgacaaaac gtgtgttgga gttttttgtg
     7261 tgctttgtca tcaactgagc cagtaggtcg agggtgccta atgagtgagc taactcacat
     7321 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     7381 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     7441 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     7501 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     7561 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     7621 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     7681 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     7741 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     7801 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     7861 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     7921 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     7981 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     8041 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     8101 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     8161 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     8221 cggggattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     8281 tcaatctaaa gtatatatga gtaaacttgg tctgacag
//
