LOCUS       sequence-366627-        8334 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-2 vector encoding PTtgO2-driven SEAP-p2A-iRFP670, PmPGK1-driven
            TtgA and PmPGK1-driven mRuby2 expression 
            (PTtgO2-CMVmin-2-SEAP-p2A-iRFP670-pA::PmPGK1-TtgA-pA::PmPGK1-mRuby2-
            pA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8334)
  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 8334)
  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: pVH666
FEATURES             Location/Qualifiers
     source          1..8334
                     /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
     primer_bind     1112..1136
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     1188..1207
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1188..1206
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2837..2893
                     /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             2900..3829
                     /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(3890..3907)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3896..4120
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4168..4667
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             5495..5728
                     /codon_start=1
                     /gene="UL48"
                     /product="transcriptional activation domain of herpes 
                     simplex virus protein VP16 (Triezenberg et al., 1988; 
                     Cousens et al., 1989)"
                     /label=VP16 AD
                     /translation="APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGF
                     TPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(5850..5866)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5850..5866)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5906..6405
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             6470..7177
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHM
                     ALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVA
                     KFAGLGGGMDELYK"
     protein_bind    7317..7338
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7455..7472)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7626..8214)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7706..7725)
                     /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 tggtaccgta tttacaaaca accatgaatg taagtatcat actctcgagt atgtcgaggt
     1081 ggcgtgtacg gtgggaggcc tatataagca gagctcgttt agtgaaccgt cagatcgcga
     1141 gctctctggc taactagaga acccactgct tactggctta tcgaaattaa tacgactcac
     1201 tatagggaga cccaagctgt tcgaagcgga attcaccatg actagttggc tgtggctggt
     1261 gctgctgctg ctgtgcctgg ccggcaacgt gcaggccgct agtatcatcc ccgtggagga
     1321 ggagaacccc gacttctgga acagagaggc cgccgaggcc ctgggcgccg ccaagaagct
     1381 gcagcccgcc cagaccgccg ccaagaacct gatcatcttc ctgggcgacg gcatgggcgt
     1441 gagcaccgtg accgccgcca gaatcctgaa gggccagaag aaggacaagc tgggccccga
     1501 gatccccctg gccatggaca gattccccta cgtggccctg agcaagacct acaacgtgga
     1561 caagcacgtg cccgacagcg gcgccaccgc caccgcctac ctgtgcggcg tgaagggcaa
     1621 cttccagacc atcggcctga gcgccgccgc cagattcaac cagtgcaaca ccaccagagg
     1681 caacgaggtg atcagcgtga tgaacagagc caagaaggcc ggcaagagcg tgggcgtggt
     1741 gaccaccacc agagtgcagc acgccagccc cgccggcacc tacgcccaca ccgtgaacag
     1801 aaactggtac agcgacgccg acgtgcccgc cagcgccaga caggagggct gccaggacat
     1861 cgccacccag ctgatcagca acatggacat cgacgtgatc ctgggcggcg gcagaaagta
     1921 catgttcaga atgggcaccc ccgaccccga gtaccccgac gactacagcc agggcggcac
     1981 cagactggac ggcaagaacc tggtgcagga gtggctggcc aagagacagg gcgccagata
     2041 cgtgtggaac agaaccgagc tgatgcaggc cagcctggac cccagcgtga cccacctgat
     2101 gggcctgttc gagcccggcg acatgaagta cgagatccac agagacagca ccctggaccc
     2161 cagcctgatg gagatgaccg aggccgccct gagactgctg agcagaaacc ccagaggctt
     2221 cttcctgttc gtggagggcg gcagaatcga ccacggccac cacgagagca gagcctacag
     2281 agccctgacc gagaccatca tgttcgacga cgccatcgag agagccggcc agctgaccag
     2341 cgaggaggac accctgagcc tggtgaccgc cgaccacagc cacgtgttca gcttcggcgg
     2401 ctaccccctg agaggcagca gcatcttcgg cctggccccc ggcaaggcca gagacagaaa
     2461 ggcctacacc gtgctgctgt acggcaacgg ccccggctac gtgctgaagg acggcgccag
     2521 acccgacgtg accgagagcg agagcggcag ccccgagtac agacagcaga gcgccgtgcc
     2581 cctggacgag gagacccacg ccggcgagga cgtggccgtg ttcgccagag gcccccaggc
     2641 ccacctggtg cacggcgtgc aggagcagac cttcatcgcc cacgtgatgg ccttcgccgc
     2701 ctgcctggag ccctacaccg cctgcgacct ggcccccccc gccggcacca ccgacgccgc
     2761 ccaccccggc tacagcagag tgggcgccgc cggcagattc gagcagaccg ctagtggtgg
     2821 ttctggtgga agcggagcta ctaacttcag cctgctgaag caggctggtg acgtggagga
     2881 gaaccctgga cctgctagtg cgcgtaaggt cgatctcacc tcctgcgatc gcgagccgat
     2941 ccacatcccc ggcagcattc agccgtgcgg ctgcctgctt gcctgcgacg cgcaggcggt
     3001 gcggatcacg cgcattacgg aaaatgccgg cgcgttcttt ggacgcgaaa ctccgcgggt
     3061 cggtgagcta ctcgccgatt acttcggcga gaccgaagcc catgcgctgc gcaacgcact
     3121 ggcgcagtcc tccgatccaa agcgaccggc gctgatcttc ggttggcgcg acggcctgac
     3181 cggccgcacc ttcgacatct cactgcatcg ccatgacggt acatcgatca tcgagttcga
     3241 gcctgcggcg gccgaacagg ccgacaatcc gctgcggctg acgcggcaga tcatcgcgcg
     3301 caccaaagaa ctgaagtcgc tcgaagagat ggccgcacgg gtgccgcgct atctgcaggc
     3361 gatgctcggc tatcaccgcg tgatgttgta ccgcttcgcg gacgacggct ccgggatggt
     3421 gatcggcgag gcgaagcgca gcgacctcga gagctttctc ggtcagcact ttccggcgtc
     3481 gctggtcccg cagcaggcgc ggctactgta cttgaagaac gcgatccgcg tggtctcgga
     3541 ttcgcgcggc atcagcagcc ggatcgtgcc cgagcacgac gcctccggcg ccgcgctcga
     3601 tctgtcgttc gcgcacctgc gcagcatctc gccctgccat ctcgaatttc tgcggaacat
     3661 gggcgtcagc gcctcgatgt cgctgtcgat catcattgac ggcacgctat ggggattgat
     3721 catctgtcat cattacgagc cgcgtgccgt gccgatggcg cagcgcgtcg cggccgaaat
     3781 gttcgccgac ttcttatcgc tgcacttcac cgccgcccac caccaacgcg ctagcggatc
     3841 caccggtgtc tagaaagctt tgaggccggc ctgcaggccc gctgatcagc ctcgactgtg
     3901 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     3961 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     4021 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     4081 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg tagcttagga ggcggaaaga
     4141 accagcgcgt ggtaccctcg agctaccggg taggggaggc gcttttccca aggcagtctg
     4201 gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc
     4261 gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg
     4321 cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt
     4381 gcaggacgtg acaaatggaa gtagcacgtc tcagtagtct cgtgcagatg gacagcaccg
     4441 ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg
     4501 ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca
     4561 ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc
     4621 gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgacc tccagcccga
     4681 gctcggtata ggttcgaagc ggaattccac catggtccgt cgaaccaaag aagaagccca
     4741 ggaaacccgt gcccagatca tcgaggcggc ggaaagggcc ttctacaagc gcggggttgc
     4801 gcgaaccacc ctggccgaca tcgccgaact ggcgggtgtg acgcgggggg cgatctactg
     4861 gcacttcaac aacaaggccg agctggtgca ggcgttgctc gacagcctgc acgagaccca
     4921 tgaccatttg gcgcgggcga gcgaaagcga ggatgaagtc gacccgcttg gctgcatgcg
     4981 caagctgctg ttgcaagtgt tcaacgagct ggtgctcgat gcccgaaccc gtcgtatcaa
     5041 tgaaatcctg catcacaagt gcgagttcac cgatgacatg tgtgaaattc gccagcagcg
     5101 ccagagtgcg gtgctggatt gccacaaggg catcaccctg gcgctggcca atgccgtacg
     5161 ccggggccag ttgcctggcg agctggatgc cgagcgagcg gcggtggcca tgtttgccta
     5221 tgtcgatggc ctgatcaggc gctggctatt gctgcctgac agtgtcgatc tgttgggtga
     5281 tgtcgaaaaa tgggtcgaca ccgggctgga tatgctgcgc ttgagcccgg ctctgcgcaa
     5341 atccgcgtac agccgcgcgc gtacgaaaaa caattacggg tctaccatcg agggcctgct
     5401 cgatctcccg gacgacgacg cccccgaaga ggcggggctg gcggctccgc gcctgtcctt
     5461 tctccccgcg ggacacacgc gcagactgtc gacggccccc ccgaccgatg tcagcctggg
     5521 ggacgagctc cacttagacg gcgaggacgt ggcgatggcg catgccgacg cgctagacga
     5581 tttcgatctg gacatgttgg gggacgggga ttccccgggt ccgggattta ccccccacga
     5641 ctccgccccc tacggcgctc tggatatggc cgacttcgag tttgagcaga tgtttaccga
     5701 tgcccttgga attgacgagt acggtggggc tagcggatcc accggtgtct agaaagctat
     5761 acaggtgcac cgctttataa taaaactgat acagagaacg ttatcatgtg tgttggtttt
     5821 ttttgtgtgt gcggtttaag ggagcctcgg tcatagctgt ttcctgtgta aaacgacggc
     5881 cagcgcgtgg taccctcgag ctaccgggta ggggaggcgc ttttcccaag gcagtctgga
     5941 gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc
     6001 acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg
     6061 ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc
     6121 aggacgtgac aaatggaagt agcacgtctc agtagtctcg tgcagatgga cagcaccgct
     6181 gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct
     6241 ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg
     6301 ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc
     6361 acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcgacctc cagcccgagc
     6421 tcggtatagg ttcgaagcgg aattcaccat gactagtggt ggttctggtg tgtctaaggg
     6481 cgaagagctg atcaaggaaa atatgcgtat gaaggtggtc atggaaggtt cggtcaacgg
     6541 ccaccaattc aaatgcacag gtgaaggaga aggcaatccg tacatgggaa ctcaaaccat
     6601 gaggatcaaa gtcatcgagg gaggacccct gccatttgcc tttgacattc ttgccacgtc
     6661 gttcatgtat ggcagccgta cttttatcaa gtacccgaaa ggcattcctg atttctttaa
     6721 acagtccttt cctgagggtt ttacttggga aagagttacg agatacgaag atggtggagt
     6781 cgtcaccgtc atgcaggaca ccagccttga ggatggctgt ctcgtttacc acgtccaagt
     6841 cagaggggta aactttccct ccaatggtcc cgtgatgcag aagaagacca agggttggga
     6901 gcctaataca gagatgatgt atccagcaga tggtggtctg aggggataca ctcatatggc
     6961 actgaaagtt gatggtggtg gccatctgtc ttgctctttc gtaacaactt acaggtcaaa
     7021 aaagaccgtc gggaacatca agatgcccgg tatccatgcc gttgatcacc gcctggaaag
     7081 gttagaggaa agtgacaatg aaatgttcgt agtacaacgc gaacacgcag ttgccaagtt
     7141 cgccgggctt ggtggtggga tggacgagct gtacaaggct agcggatcca ccggtgtcta
     7201 gaaagctcag aaattatgag agcgtttaat aaagtattga ctatggttga caaaacgtgt
     7261 gttggagttt tttgtgtgct ttgtcatcaa ctgagccagt aggtcgaggg tgcctaatga
     7321 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     7381 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     7441 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     7501 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     7561 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     7621 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     7681 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     7741 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     7801 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     7861 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     7921 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     7981 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     8041 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     8101 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     8161 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     8221 cctttgatct tttctacggg gattatcaaa aaggatcttc acctagatcc ttttaaatta
     8281 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acag
//
