LOCUS       sequence-365625-        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::A2-pA::spacer-PmPGK1-IgkSS-Nluc-p2A-mTag
            BFP2-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: pTS2320
FEATURES             Location/Qualifiers
     source          1..8318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(68..859,860..928))
                     /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     691..710
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(929..1033)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1103..1482
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1483..1686
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1637..1657
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1731..1750
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1731..1749
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3368..3424
                     /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             3431..4360
                     /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(4421..4438)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4427..4651
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4797..4813)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4797..4813)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4945..4966)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5157..5176
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5276..5775
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5311..5334)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5708..5727
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     5828..5884
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             5906..6418
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             6446..6502
                     /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             6521..7228
                     /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    7368..7389
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7506..7523)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7677..8265)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7757..7776)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
       61 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
      121 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
      181 ctggccccag tgctgcaatg ataccgcggc tcccacgctc accggctcca gatttatcag
      241 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
      301 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
      361 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
      421 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
      481 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
      541 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
      601 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
      661 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
      721 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
      781 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
      841 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
      901 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
      961 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     1021 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accaagatct
     1081 ccacgcgtgg taccctcgag ttgacattga ttattgagta gttattaata gtaatcaatt
     1141 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     1201 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     1261 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     1321 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     1381 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     1441 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     1501 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     1561 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     1621 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     1681 agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact
     1741 cactataggg agacccaagc tgttcgaagc ggaattcacc atgactagtc tgctgctgct
     1801 gctgctgggc ctgagactgc agctgagcct gggcatcatc cccgtggagg aggagaaccc
     1861 cgacttctgg aacagagagg ccgccgaggc cctgggcgcc gccaagaagc tgcagcccgc
     1921 ccagaccgcc gccaagaacc tgatcatctt cctgggcgac ggcatgggcg tgagcaccgt
     1981 gaccgccgcc agaatcctga agggccagaa gaaggacaag ctgggccccg agatccccct
     2041 ggccatggac agattcccct acgtggccct gagcaagacc tacaacgtgg acaagcacgt
     2101 gcccgacagc ggcgccaccg ccaccgccta cctgtgcggc gtgaagggca acttccagac
     2161 catcggcctg agcgccgccg ccagattcaa ccagtgcaac accaccagag gcaacgaggt
     2221 gatcagcgtg atgaacagag ccaagaaggc cggcaagagc gtgggcgtgg tgaccaccac
     2281 cagagtgcag cacgccagcc ccgccggcac ctacgcccac accgtgaaca gaaactggta
     2341 cagcgacgcc gacgtgcccg ccagcgccag acaggagggc tgccaggaca tcgccaccca
     2401 gctgatcagc aacatggaca tcgacgtgat cctgggcggc ggcagaaagt acatgttcag
     2461 aatgggcacc cccgaccccg agtaccccga cgactacagc cagggcggca ccagactgga
     2521 cggcaagaac ctggtgcagg agtggctggc caagagacag ggcgccagat acgtgtggaa
     2581 cagaaccgag ctgatgcagg ccagcctgga ccccagcgtg acccacctga tgggcctgtt
     2641 cgagcccggc gacatgaagt acgagatcca cagagacagc accctggacc ccagcctgat
     2701 ggagatgacc gaggccgccc tgagactgct gagcagaaac cccagaggct tcttcctgtt
     2761 cgtggagggc ggcagaatcg accacggcca ccacgagagc agagcctaca gagccctgac
     2821 cgagaccatc atgttcgacg acgccatcga gagagccggc cagctgacca gcgaggagga
     2881 caccctgagc ctggtgaccg ccgaccacag ccacgtgttc agcttcggcg gctaccccct
     2941 gagaggcagc agcatcttcg gcctggcccc cggcaaggcc agagacagaa aggcctacac
     3001 cgtgctgctg tacggcaacg gccccggcta cgtgctgaag gacggcgcca gacccgacgt
     3061 gaccgagagc gagagcggca gccccgagta cagacagcag agcgccgtgc ccctggacga
     3121 ggagacccac gccggcgagg acgtggccgt gttcgccaga ggcccccagg cccacctggt
     3181 gcacggcgtg caggagcaga ccttcatcgc ccacgtgatg gccttcgccg cctgcctgga
     3241 gccctacacc gcctgcgacc tggccccccc cgccggcacc accgacgccg cccaccccgg
     3301 ctacagcaga gtgggcgccg ccggcagatt cgagcagacc gctagtggtg gttctggtgg
     3361 aagcggagct actaacttca gcctgctgaa gcaggctggt gacgtggagg agaaccctgg
     3421 acctgctagt gcgcgtaagg tcgatctcac ctcctgcgat cgcgagccga tccacatccc
     3481 cggcagcatt cagccgtgcg gctgcctgct tgcctgcgac gcgcaggcgg tgcggatcac
     3541 gcgcattacg gaaaatgccg gcgcgttctt tggacgcgaa actccgcggg tcggtgagct
     3601 actcgccgat tacttcggcg agaccgaagc ccatgcgctg cgcaacgcac tggcgcagtc
     3661 ctccgatcca aagcgaccgg cgctgatctt cggttggcgc gacggcctga ccggccgcac
     3721 cttcgacatc tcactgcatc gccatgacgg tacatcgatc atcgagttcg agcctgcggc
     3781 ggccgaacag gccgacaatc cgctgcggct gacgcggcag atcatcgcgc gcaccaaaga
     3841 actgaagtcg ctcgaagaga tggccgcacg ggtgccgcgc tatctgcagg cgatgctcgg
     3901 ctatcaccgc gtgatgttgt accgcttcgc ggacgacggc tccgggatgg tgatcggcga
     3961 ggcgaagcgc agcgacctcg agagctttct cggtcagcac tttccggcgt cgctggtccc
     4021 gcagcaggcg cggctactgt acttgaagaa cgcgatccgc gtggtctcgg attcgcgcgg
     4081 catcagcagc cggatcgtgc ccgagcacga cgcctccggc gccgcgctcg atctgtcgtt
     4141 cgcgcacctg cgcagcatct cgccctgcca tctcgaattt ctgcggaaca tgggcgtcag
     4201 cgcctcgatg tcgctgtcga tcatcattga cggcacgcta tggggattga tcatctgtca
     4261 tcattacgag ccgcgtgccg tgccgatggc gcagcgcgtc gcggccgaaa tgttcgccga
     4321 cttcttatcg ctgcacttca ccgccgccca ccaccaacgc gctagcggat ccaccggtgt
     4381 ctagaaagct ttgaggccgg cctgcaggcc cgctgatcag cctcgactgt gccttctagt
     4441 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     4501 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     4561 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     4621 aggcatgctg gggatgcggt gggctctatg gtagcttagg aggcggaaag aaccagcgcg
     4681 ggagaccgtc agttggtctc cagctataca ggtgcaccgc tttataataa aactgataca
     4741 gagaacgtta tcatgtgtgt tggttttttt tgtgtgtgcg gtttaaggga gcctcggtca
     4801 tagctgtttc ctgtgtaaaa cgacggccag cgcgtattcg cgttaaattt ttgttaaatc
     4861 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     4921 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     4981 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     5041 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     5101 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     5161 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     5221 accaccacac ccgccgcgct taatgcgccg ctacaggggg taccctcgag ctaccgggta
     5281 ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact
     5341 tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa
     5401 ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag
     5461 ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc
     5521 agtagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc
     5581 agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg
     5641 tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga
     5701 ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca
     5761 tctccgggcc tttcgacctc cagcccgagc tcggtatagg ttcgaagcgg aattcaccat
     5821 gactagtgag acagacacac tcctgctatg ggtactgctg ctctgggttc caggttccac
     5881 tggtgacgct agtggtggtt ctggtatggt cttcacactc gaagatttcg ttggggactg
     5941 gcgacagaca gccggctaca acctggacca agtccttgaa cagggaggtg tgtccagttt
     6001 gtttcagaat ctcggggtgt ccgtaactcc gatccaaagg attgtcctga gcggtgaaaa
     6061 tgggctgaag atcgacatcc atgtcatcat cccgtatgaa ggtctgagcg gcgaccaaat
     6121 gggccagatc gaaaaaattt ttaaggtggt gtaccctgtg gatgatcatc actttaaggt
     6181 gatcctgcac tatggcacac tggtaatcga cggggttacg ccgaacatga tcgactattt
     6241 cggacggccg tatgaaggca tcgccgtgtt cgacggcaaa aagatcactg taacagggac
     6301 cctgtggaac ggcaacaaaa ttatcgacga gcgcctgatc aaccccgacg gctccctgct
     6361 gttccgagta accatcaacg gagtgaccgg ctggcggctg tgcgaacgca ttctggcggc
     6421 tagtggtggt tctggtggaa gcggagctac taacttcagc ctgctgaagc aggctggtga
     6481 cgtggaggag aaccctggac ctgctagtgg tggttctggt gtgtctaagg gcgaagagct
     6541 gattaaggag aacatgcaca tgaagctgta catggagggc accgtggaca accatcactt
     6601 caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa
     6661 ggtggtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacta gcttcctcta
     6721 cggcagcaag accttcatca accacaccca gggcatcccc gacttcttca agcagtcctt
     6781 ccctgagggc ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc
     6841 tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaaga tcagaggggt
     6901 gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggccttcac
     6961 cgagacgctg taccccgctg acggcggcct ggaaggcaga aacgacatgg ccctgaagct
     7021 cgtgggcggg agccatctga tcgcaaacgc caagaccaca tatagatcca agaaacccgc
     7081 taagaacctc aagatgcctg gcgtctacta tgtggactac agactggaaa gaatcaagga
     7141 ggccaacaac gagacctacg tcgagcagca cgaggtggca gtggccagat actgcgacct
     7201 ccctagcaaa ctggggcaca aacttaatgc tagcggatcc accggtgtct agaaagctca
     7261 gaaattatga gagcgtttaa taaagtattg actatggttg acaaaacgtg tgttggagtt
     7321 ttttgtgtgc tttgtcatca actgagccag taggtcgagg gtgcctaatg agtgagctaa
     7381 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     7441 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     7501 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     7561 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     7621 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     7681 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     7741 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     7801 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     7861 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     7921 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     7981 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     8041 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     8101 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     8161 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     8221 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     8281 ttttctacgg ggattatcaa aaaggatctt cacctaga
//
