LOCUS       sequence-372400-        8049 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' 
            insulator sequence derived from the pGL3 plasmid 
            (PhCMV-SEAP-p2A-iRFP670-pA::pGL3-PmPGK1-IgkSS-Nluc-p2A-mTagBFP2-pA::
            A3-pA). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8049)
  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 8049)
  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: pTS2326
FEATURES             Location/Qualifiers
     source          1..8049
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(67..858,859..927))
                     /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     690..709
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(928..1032)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1102..1481
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1482..1685
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1636..1656
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1730..1749
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1730..1748
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3367..3423
                     /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             3430..4359
                     /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(4420..4437)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4426..4650
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4681..4729
                     /note="synthetic polyadenylation signal"
     misc_feature    4743..4834
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4783..4802
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        4852..5351
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4887..4910)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     5284..5303
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     sig_peptide     5404..5460
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="ETDTLLLWVLLLWVPGSTG"
     CDS             5482..5994
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             6022..6078
                     /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             6097..6804
                     /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(6926..6942)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6926..6942)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    7098..7119
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7236..7253)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7407..7995)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7487..7506)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
       61 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
      121 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
      181 tggccccagt gctgcaatga taccgcggct cccacgctca ccggctccag atttatcagc
      241 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
      301 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
      361 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
      421 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
      481 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
      541 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
      601 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
      661 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
      721 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
      781 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
      841 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
      901 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
      961 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     1021 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccaagatctc
     1081 cacgcgtggt accctcgagt tgacattgat tattgagtag ttattaatag taatcaatta
     1141 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1201 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1261 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1321 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1381 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1441 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1501 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1561 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1621 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1681 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     1741 actataggga gacccaagct gttcgaagcg gaattcacca tgactagtct gctgctgctg
     1801 ctgctgggcc tgagactgca gctgagcctg ggcatcatcc ccgtggagga ggagaacccc
     1861 gacttctgga acagagaggc cgccgaggcc ctgggcgccg ccaagaagct gcagcccgcc
     1921 cagaccgccg ccaagaacct gatcatcttc ctgggcgacg gcatgggcgt gagcaccgtg
     1981 accgccgcca gaatcctgaa gggccagaag aaggacaagc tgggccccga gatccccctg
     2041 gccatggaca gattccccta cgtggccctg agcaagacct acaacgtgga caagcacgtg
     2101 cccgacagcg gcgccaccgc caccgcctac ctgtgcggcg tgaagggcaa cttccagacc
     2161 atcggcctga gcgccgccgc cagattcaac cagtgcaaca ccaccagagg caacgaggtg
     2221 atcagcgtga tgaacagagc caagaaggcc ggcaagagcg tgggcgtggt gaccaccacc
     2281 agagtgcagc acgccagccc cgccggcacc tacgcccaca ccgtgaacag aaactggtac
     2341 agcgacgccg acgtgcccgc cagcgccaga caggagggct gccaggacat cgccacccag
     2401 ctgatcagca acatggacat cgacgtgatc ctgggcggcg gcagaaagta catgttcaga
     2461 atgggcaccc ccgaccccga gtaccccgac gactacagcc agggcggcac cagactggac
     2521 ggcaagaacc tggtgcagga gtggctggcc aagagacagg gcgccagata cgtgtggaac
     2581 agaaccgagc tgatgcaggc cagcctggac cccagcgtga cccacctgat gggcctgttc
     2641 gagcccggcg acatgaagta cgagatccac agagacagca ccctggaccc cagcctgatg
     2701 gagatgaccg aggccgccct gagactgctg agcagaaacc ccagaggctt cttcctgttc
     2761 gtggagggcg gcagaatcga ccacggccac cacgagagca gagcctacag agccctgacc
     2821 gagaccatca tgttcgacga cgccatcgag agagccggcc agctgaccag cgaggaggac
     2881 accctgagcc tggtgaccgc cgaccacagc cacgtgttca gcttcggcgg ctaccccctg
     2941 agaggcagca gcatcttcgg cctggccccc ggcaaggcca gagacagaaa ggcctacacc
     3001 gtgctgctgt acggcaacgg ccccggctac gtgctgaagg acggcgccag acccgacgtg
     3061 accgagagcg agagcggcag ccccgagtac agacagcaga gcgccgtgcc cctggacgag
     3121 gagacccacg ccggcgagga cgtggccgtg ttcgccagag gcccccaggc ccacctggtg
     3181 cacggcgtgc aggagcagac cttcatcgcc cacgtgatgg ccttcgccgc ctgcctggag
     3241 ccctacaccg cctgcgacct ggcccccccc gccggcacca ccgacgccgc ccaccccggc
     3301 tacagcagag tgggcgccgc cggcagattc gagcagaccg ctagtggtgg ttctggtgga
     3361 agcggagcta ctaacttcag cctgctgaag caggctggtg acgtggagga gaaccctgga
     3421 cctgctagtg cgcgtaaggt cgatctcacc tcctgcgatc gcgagccgat ccacatcccc
     3481 ggcagcattc agccgtgcgg ctgcctgctt gcctgcgacg cgcaggcggt gcggatcacg
     3541 cgcattacgg aaaatgccgg cgcgttcttt ggacgcgaaa ctccgcgggt cggtgagcta
     3601 ctcgccgatt acttcggcga gaccgaagcc catgcgctgc gcaacgcact ggcgcagtcc
     3661 tccgatccaa agcgaccggc gctgatcttc ggttggcgcg acggcctgac cggccgcacc
     3721 ttcgacatct cactgcatcg ccatgacggt acatcgatca tcgagttcga gcctgcggcg
     3781 gccgaacagg ccgacaatcc gctgcggctg acgcggcaga tcatcgcgcg caccaaagaa
     3841 ctgaagtcgc tcgaagagat ggccgcacgg gtgccgcgct atctgcaggc gatgctcggc
     3901 tatcaccgcg tgatgttgta ccgcttcgcg gacgacggct ccgggatggt gatcggcgag
     3961 gcgaagcgca gcgacctcga gagctttctc ggtcagcact ttccggcgtc gctggtcccg
     4021 cagcaggcgc ggctactgta cttgaagaac gcgatccgcg tggtctcgga ttcgcgcggc
     4081 atcagcagcc ggatcgtgcc cgagcacgac gcctccggcg ccgcgctcga tctgtcgttc
     4141 gcgcacctgc gcagcatctc gccctgccat ctcgaatttc tgcggaacat gggcgtcagc
     4201 gcctcgatgt cgctgtcgat catcattgac ggcacgctat ggggattgat catctgtcat
     4261 cattacgagc cgcgtgccgt gccgatggcg cagcgcgtcg cggccgaaat gttcgccgac
     4321 ttcttatcgc tgcacttcac cgccgcccac caccaacgcg ctagcggatc caccggtgtc
     4381 tagaaagctt tgaggccggc ctgcaggccc gctgatcagc ctcgactgtg ccttctagtt
     4441 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     4501 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     4561 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     4621 ggcatgctgg ggatgcggtg ggctctatgg tagcttagga ggcggaaaga accagcgcgt
     4681 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta
     4741 ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc
     4801 cccagtgcaa gtgcaggtgc cagaacattt ctctggtacc ctcgagctac cgggtagggg
     4861 aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
     4921 gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg
     4981 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
     5041 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcagta
     5101 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
     5161 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
     5221 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     5281 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     5341 cgggcctttc gacctccagc ccgagctcgg tataggttcg aagcggaatt caccatgact
     5401 agtgagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt
     5461 gacgctagtg gtggttctgg tatggtcttc acactcgaag atttcgttgg ggactggcga
     5521 cagacagccg gctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgttt
     5581 cagaatctcg gggtgtccgt aactccgatc caaaggattg tcctgagcgg tgaaaatggg
     5641 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcggcga ccaaatgggc
     5701 cagatcgaaa aaatttttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc
     5761 ctgcactatg gcacactggt aatcgacggg gttacgccga acatgatcga ctatttcgga
     5821 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg
     5881 tggaacggca acaaaattat cgacgagcgc ctgatcaacc ccgacggctc cctgctgttc
     5941 cgagtaacca tcaacggagt gaccggctgg cggctgtgcg aacgcattct ggcggctagt
     6001 ggtggttctg gtggaagcgg agctactaac ttcagcctgc tgaagcaggc tggtgacgtg
     6061 gaggagaacc ctggacctgc tagtggtggt tctggtgtgt ctaagggcga agagctgatt
     6121 aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag
     6181 tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg
     6241 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc
     6301 agcaagacct tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct
     6361 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
     6421 caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac
     6481 ttcacatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgag
     6541 acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg
     6601 ggcgggagcc atctgatcgc aaacgccaag accacatata gatccaagaa acccgctaag
     6661 aacctcaaga tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc
     6721 aacaacgaga cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct
     6781 agcaaactgg ggcacaaact taatgctagc ggatccaccg gtgtctagaa agctatacag
     6841 gtgcaccgct ttataataaa actgatacag agaacgttat catgtgtgtt ggtttttttt
     6901 gtgtgtgcgg tttaagggag cctcggtcat agctgtttcc tgtgtaaaac gacggccagc
     6961 gcgggagacg gtcagttcgt ctccagctca gaaattatga gagcgtttaa taaagtattg
     7021 actatggttg acaaaacgtg tgttggagtt ttttgtgtgc tttgtcatca actgagccag
     7081 taggtcgagg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     7141 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     7201 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     7261 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     7321 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     7381 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     7441 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     7501 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     7561 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     7621 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     7681 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     7741 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     7801 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     7861 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     7921 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     7981 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggattatcaa aaaggatctt
     8041 cacctagat
//
