LOCUS       sequence-366623-        8298 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-2 vector encoding PTetO7-driven SEAP-p2A-iRFP670, PmPGK1-driven
            rtTA and PmPGK1-driven YPet expression 
            (PTetO7-CMVmin-2-SEAP-p2A-iRFP670-pA::PmPGK1-rtTA-pA::PmPGK1-YPet-pA
            )..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8298)
  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 8298)
  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: pVH664
FEATURES             Location/Qualifiers
     source          1..8298
                     /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
     protein_bind    1047..1065
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1083..1101
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1119..1137
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1155..1173
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1191..1209
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1227..1245
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1263..1281
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1335..1359
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     1411..1430
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1411..1429
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3060..3116
                     /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             3123..4052
                     /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(4113..4130)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4119..4343
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4391..4890
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             4946..5686
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="SRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTD
                     SMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPAD
                     ALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(5808..5824)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5808..5824)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5864..6363
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6731..6750)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    7281..7302
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7419..7436)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7590..8178)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7670..7689)
                     /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 tccgtacacg cctaaagcat atacgttctc tatcactgat agggagtaaa ctggatatac
     1081 gttctctatc actgataggg agtaaactgt agatacgttc tctatcactg atagggagta
     1141 aactggtcat acgttctcta tcactgatag ggagtaaact ccttatacgt tctctatcac
     1201 tgatagggag taaagtctgc atacgttctc tatcactgat agggagtaaa ctcttcatac
     1261 gttctctatc actgataggg agtaaactcg agtatgtcga ggtggcgtgt acggtgggag
     1321 gcctatataa gcagagctcg tttagtgaac cgtcagatcg cgagctctct ggctaactag
     1381 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
     1441 tgttcgaagc ggaattcacc atgactagtt ggctgtggct ggtgctgctg ctgctgtgcc
     1501 tggccggcaa cgtgcaggcc gctagtatca tccccgtgga ggaggagaac cccgacttct
     1561 ggaacagaga ggccgccgag gccctgggcg ccgccaagaa gctgcagccc gcccagaccg
     1621 ccgccaagaa cctgatcatc ttcctgggcg acggcatggg cgtgagcacc gtgaccgccg
     1681 ccagaatcct gaagggccag aagaaggaca agctgggccc cgagatcccc ctggccatgg
     1741 acagattccc ctacgtggcc ctgagcaaga cctacaacgt ggacaagcac gtgcccgaca
     1801 gcggcgccac cgccaccgcc tacctgtgcg gcgtgaaggg caacttccag accatcggcc
     1861 tgagcgccgc cgccagattc aaccagtgca acaccaccag aggcaacgag gtgatcagcg
     1921 tgatgaacag agccaagaag gccggcaaga gcgtgggcgt ggtgaccacc accagagtgc
     1981 agcacgccag ccccgccggc acctacgccc acaccgtgaa cagaaactgg tacagcgacg
     2041 ccgacgtgcc cgccagcgcc agacaggagg gctgccagga catcgccacc cagctgatca
     2101 gcaacatgga catcgacgtg atcctgggcg gcggcagaaa gtacatgttc agaatgggca
     2161 cccccgaccc cgagtacccc gacgactaca gccagggcgg caccagactg gacggcaaga
     2221 acctggtgca ggagtggctg gccaagagac agggcgccag atacgtgtgg aacagaaccg
     2281 agctgatgca ggccagcctg gaccccagcg tgacccacct gatgggcctg ttcgagcccg
     2341 gcgacatgaa gtacgagatc cacagagaca gcaccctgga ccccagcctg atggagatga
     2401 ccgaggccgc cctgagactg ctgagcagaa accccagagg cttcttcctg ttcgtggagg
     2461 gcggcagaat cgaccacggc caccacgaga gcagagccta cagagccctg accgagacca
     2521 tcatgttcga cgacgccatc gagagagccg gccagctgac cagcgaggag gacaccctga
     2581 gcctggtgac cgccgaccac agccacgtgt tcagcttcgg cggctacccc ctgagaggca
     2641 gcagcatctt cggcctggcc cccggcaagg ccagagacag aaaggcctac accgtgctgc
     2701 tgtacggcaa cggccccggc tacgtgctga aggacggcgc cagacccgac gtgaccgaga
     2761 gcgagagcgg cagccccgag tacagacagc agagcgccgt gcccctggac gaggagaccc
     2821 acgccggcga ggacgtggcc gtgttcgcca gaggccccca ggcccacctg gtgcacggcg
     2881 tgcaggagca gaccttcatc gcccacgtga tggccttcgc cgcctgcctg gagccctaca
     2941 ccgcctgcga cctggccccc cccgccggca ccaccgacgc cgcccacccc ggctacagca
     3001 gagtgggcgc cgccggcaga ttcgagcaga ccgctagtgg tggttctggt ggaagcggag
     3061 ctactaactt cagcctgctg aagcaggctg gtgacgtgga ggagaaccct ggacctgcta
     3121 gtgcgcgtaa ggtcgatctc acctcctgcg atcgcgagcc gatccacatc cccggcagca
     3181 ttcagccgtg cggctgcctg cttgcctgcg acgcgcaggc ggtgcggatc acgcgcatta
     3241 cggaaaatgc cggcgcgttc tttggacgcg aaactccgcg ggtcggtgag ctactcgccg
     3301 attacttcgg cgagaccgaa gcccatgcgc tgcgcaacgc actggcgcag tcctccgatc
     3361 caaagcgacc ggcgctgatc ttcggttggc gcgacggcct gaccggccgc accttcgaca
     3421 tctcactgca tcgccatgac ggtacatcga tcatcgagtt cgagcctgcg gcggccgaac
     3481 aggccgacaa tccgctgcgg ctgacgcggc agatcatcgc gcgcaccaaa gaactgaagt
     3541 cgctcgaaga gatggccgca cgggtgccgc gctatctgca ggcgatgctc ggctatcacc
     3601 gcgtgatgtt gtaccgcttc gcggacgacg gctccgggat ggtgatcggc gaggcgaagc
     3661 gcagcgacct cgagagcttt ctcggtcagc actttccggc gtcgctggtc ccgcagcagg
     3721 cgcggctact gtacttgaag aacgcgatcc gcgtggtctc ggattcgcgc ggcatcagca
     3781 gccggatcgt gcccgagcac gacgcctccg gcgccgcgct cgatctgtcg ttcgcgcacc
     3841 tgcgcagcat ctcgccctgc catctcgaat ttctgcggaa catgggcgtc agcgcctcga
     3901 tgtcgctgtc gatcatcatt gacggcacgc tatggggatt gatcatctgt catcattacg
     3961 agccgcgtgc cgtgccgatg gcgcagcgcg tcgcggccga aatgttcgcc gacttcttat
     4021 cgctgcactt caccgccgcc caccaccaac gcgctagcgg atccaccggt gtctagaaag
     4081 ctttgaggcc ggcctgcagg cccgctgatc agcctcgact gtgccttcta gttgccagcc
     4141 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     4201 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     4261 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     4321 tggggatgcg gtgggctcta tggtagctta ggaggcggaa agaaccagcg cgtggtaccc
     4381 tcgagctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca
     4441 gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc
     4501 accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct
     4561 cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg
     4621 gaagtagcac gtctcagtag tctcgtgcag atggacagca ccgctgagca atggaagcgg
     4681 gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg
     4741 ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc
     4801 cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg
     4861 ttctcctctt cctcatctcc gggcctttcg acctccagcc cgagctcggt ataggttcga
     4921 agcggaattc accatgacta gtggatcaag actggacaag agcaaagtca taaactctgc
     4981 tctggaatta ctcaatggag tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa
     5041 gctgggagtt gagcagccta ccctgtactg gcacgtgaag aacaagcggg ccctgctcga
     5101 tgccctgcca atcgagatgc tggacaggca tcatacccac tcctgccccc tggaaggcga
     5161 gtcatggcaa gactttctgc ggaacaacgc caagtcatac cgctgtgctc tcctctcaca
     5221 tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca gagaaacagt acgaaaccct
     5281 ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg cactgtacgc
     5341 tctgtccgcc gtgggccact ttacactggg ctgcgtattg gaggaacagg agcatcaagt
     5401 agcaaaagag gaaagagaga cacctaccac cgattctatg cccccacttc tgaaacaagc
     5461 aattgagctg ttcgaccggc agggagccga acctgccttc cttttcggcc tggaactaat
     5521 catatgtggc ctggagaaac agctaaagtg cgaaagcggc gggccgaccg acgcccttga
     5581 cgattttgac ttagacatgc tcccagccga tgcccttgac gactttgacc ttgatatgct
     5641 gcctgctgac gctcttgacg attttgacct tgacatgctc cccggggcta gcggatccac
     5701 cggtgtctag aaagctatac aggtgcaccg ctttataata aaactgatac agagaacgtt
     5761 atcatgtgtg ttggtttttt ttgtgtgtgc ggtttaaggg agcctcggtc atagctgttt
     5821 cctgtgtaaa acgacggcca gcgcgtggta ccctcgagct accgggtagg ggaggcgctt
     5881 ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca
     5941 agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc
     6001 tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc
     6061 ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcag tagtctcgtg
     6121 cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag
     6181 cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg
     6241 ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc
     6301 tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt
     6361 tcgacctcca gcccgagctc ggtataggtt cgaagcggaa ttcaccatga ctagtggtgg
     6421 ttctggtgtg agcaaaggcg aagagctgtt caccggcgtg gtgcccatcc tggtggagct
     6481 ggacggcgac gtgaacggcc acaagttcag cgtgagcggc gagggcgagg gcgacgccac
     6541 ctacggcaag ctgaccctga agctgctgtg caccaccggc aagctgcccg tgccctggcc
     6601 caccctggtg accaccctgg gctacggcgt gcagtgcttc gcccggtacc ccgaccacat
     6661 gaagcagcac gacttcttca agagcgccat gcccgagggc tacgtgcagg agcggaccat
     6721 cttcttcaag gacgacggca actacaagac ccgggccgag gtgaagttcg agggcgacac
     6781 cctggtgaac cggatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     6841 ccacaagctg gagtacaact acaacagcca caacgtgtac atcaccgccg acaagcagaa
     6901 gaacggcatc aaggccaact tcaagatccg gcacaacatc gaggacggcg gcgtgcagct
     6961 ggccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     7021 ccactacctg agctaccaga gcgccctgtt caaggacccc aacgagaagc gggaccacat
     7081 ggtgctgctg gagttcctga ccgccgccgg catcaccgag ggcatggacg agctgtacaa
     7141 ggctagcgga tccaccggtg tctagaaagc tcagaaatta tgagagcgtt taataaagta
     7201 ttgactatgg ttgacaaaac gtgtgttgga gttttttgtg tgctttgtca tcaactgagc
     7261 cagtaggtcg agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     7321 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     7381 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     7441 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     7501 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     7561 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     7621 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     7681 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     7741 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     7801 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     7861 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     7921 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     7981 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     8041 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     8101 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     8161 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggattat caaaaaggat
     8221 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     8281 gtaaacttgg tctgacag
//
