LOCUS       sequence-365639-        7770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tier-3 vector for stable integration via lentiviral transduction in 
            inverted direction with polyA carrying a constitutive expression 
            cassette for puromyine and Ypet 
            (PCMV-5'LTR-Psi-gag-env-PRPBSA-YPet-p2A-PuroR-pA::A2-pA::A3-pA-WPRE-
            3'LTR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7770)
  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 7770)
  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: pTS2333_Tier3(Lenti_inverse)-YPet_Puro
FEATURES             Location/Qualifiers
     source          1..7770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      212..800
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     701..720
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     LTR             863..1096
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    1168..1756
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1274..1285
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1683..1703
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(1930..1947)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1936..2160
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2306..2322)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2306..2322)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3315..3334)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             3753..3809
                     /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             3828..4421
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     4321..4341
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4605..4722
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(5020..5064)
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    5249..5482
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    5975..6100
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             6147..6327
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(6345..6543)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6370..6390)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6545..6848
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6896..7000
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7001..7069,7070..7770,1..91)
                     /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     complement(7219..7238)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
       61 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
      121 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
      181 taatccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
      241 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
      301 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
      361 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
      421 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
      481 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
      541 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
      601 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
      661 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
      721 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
      781 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
      841 gctggccttt tgctcacatg tctgctagag attttccaca ctgactaaaa gggtctgagg
      901 gatctctagt taccagagtc acacaacaga cgggcacaca ctacttgaag cactcaaggc
      961 aaactttatt gaggcttaag cagtgggttc cctagttagc cagagagctc ccaggctcag
     1021 atctggtcta accagagaaa cccagtacaa gcaaaaagca gatcttgtct tcgttgggag
     1081 tgaattagcc cttccagtcc ccccttttct tttaaaaagt ggctaagatc tacagctgcc
     1141 ttgtaagtca ttggtcttaa agtcgacgcg gggaggcggc ccaaagggag atccgactcg
     1201 tctgagggcg aaggcgaaga cgcggaagag gccgcagagc cggcagcagg ccgcgggaag
     1261 gaaggtccgc tggattgagg gccgaaggga cgtagcagaa ggacgtcccg cgcagaatcc
     1321 aggtggcaac acaggcgagc agccaaggaa aggacgatga tttccccgac aacaccacgg
     1381 aattgtcagt gcccaacagc cgagcccctg tccagcagcg ggcaaggcag gcggcgatga
     1441 gttccgccgt ggcaataggg agggggaaag cgaaagtccc ggaaaggagc tgacaggtgg
     1501 tggcaatgcc ccaaccagtg ggggttgcgt cagcaaacac agtgcacacc acgccacgtt
     1561 gcctgacaac gggccacaac tcctcataaa gagacagcaa ccaggattta tacaaggagg
     1621 agaaaatgaa agccatacgg gaagcaatag catgatacaa aggcattaaa gcagcgtatc
     1681 cacatagcgt aaaaggagca acatagttaa gaataccagt caatctttca caaattttgt
     1741 aatccagagg ttgattgatc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     1801 gcaacgcaat taatagctac gcgtggtacc ctcgaggagc tcttcgaagc ggaattcacc
     1861 atgactagtg ctagcggatc caccggtgtc tagaaagctt tgaggccggc ctgcaggccc
     1921 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     1981 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     2041 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2101 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     2161 tagcttagga ggcggaaaga accagcgcgg gagaccgtca gttggtctcc agctatacag
     2221 gtgcaccgct ttataataaa actgatacag agaacgttat catgtgtgtt ggtttttttt
     2281 gtgtgtgcgg tttaagggag cctcggtcat agctgtttcc tgtgtaaaac gacggccagc
     2341 gcgtggtacc ctcgagcgtg accctgtctc acaaaataaa gtaagcccgg actgagtgcg
     2401 gaaaggcggg cctggcgggt ctggactccc catgcgggcc accagaggcc ctgcagcctt
     2461 cagtcgcttg aaggggtaat ggcgcttcca ctcacaaaca tggcggacag agcgtgtgaa
     2521 cgagatgaac agcccctcaa aaatatggcc gccgaggctg gacggccgtg ccccagcagc
     2581 accgcctccg cgccccacgt gatctctcgc cgggcacagc gctgaccgcg gaggtccaac
     2641 cggaagaatg tccggattgg acattcggaa gagggcccgc cttccctggg gaatctctgc
     2701 gcacgcgcag aacgcttcga ccaatgaaaa cacaggaagc cgtccgcgca accgcgttgc
     2761 gtcacttctg ccgcccctgt ttcaaggtat atagccgtag acggaacttc gcctttctct
     2821 cggccttagc gccatttttt tgggtgagtg ttttttggtt cctgcgttgg gattccgtgt
     2881 acaatccata gacatctgac ctcggcactt agcatcatca cagcaaacta actgtagcct
     2941 ttctctcttt ccctgtagaa acctctgcac ctagggagct cgaattcacc atgactagtg
     3001 gtggttctgg tgtgagcaaa ggcgaagagc tgttcaccgg cgtggtgccc atcctggtgg
     3061 agctggacgg cgacgtgaac ggccacaagt tcagcgtgag cggcgagggc gagggcgacg
     3121 ccacctacgg caagctgacc ctgaagctgc tgtgcaccac cggcaagctg cccgtgccct
     3181 ggcccaccct ggtgaccacc ctgggctacg gcgtgcagtg cttcgcccgg taccccgacc
     3241 acatgaagca gcacgacttc ttcaagagcg ccatgcccga gggctacgtg caggagcgga
     3301 ccatcttctt caaggacgac ggcaactaca agacccgggc cgaggtgaag ttcgagggcg
     3361 acaccctggt gaaccggatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     3421 tgggccacaa gctggagtac aactacaaca gccacaacgt gtacatcacc gccgacaagc
     3481 agaagaacgg catcaaggcc aacttcaaga tccggcacaa catcgaggac ggcggcgtgc
     3541 agctggccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     3601 acaaccacta cctgagctac cagagcgccc tgttcaagga ccccaacgag aagcgggacc
     3661 acatggtgct gctggagttc ctgaccgccg ccggcatcac cgagggcatg gacgagctgt
     3721 acaaggctag tggtggttct ggtggaagcg gagctactaa cttcagcctg ctgaagcagg
     3781 ctggtgacgt ggaggagaac cctggacctg ctagtggtgg ttctggtacc gagtacaagc
     3841 ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg
     3901 cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac atcgagcggg
     3961 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     4021 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     4081 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     4141 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     4201 ccaccgtcgg cgtgtcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     4261 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagaca tccgcgcccc
     4321 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     4381 aaggaccgcg cacctggtgc atgacccgca agcccggtgc cgctagcgga tccaccggtg
     4441 tctagaaagc tcagaaatta tgagagcgtt taataaagta ttgactatgg ttgacaaaac
     4501 gtgtgttgga gttttttgtg tgctttgtca tcaactgagc cagtagcgcg tgaccaagta
     4561 tccccgcttc ttctcgcgtc actgctgtcc ctgtaataaa cccgaaaatt ttgaattttt
     4621 gtaatttgtt tttgtaattc tttagtttgt atgtctgttg ctattatgtc tactattctt
     4681 tcccctgcac tgtacccccc aatcccccct tttcttttaa aattgtggat gaatactgcc
     4741 atttgtctgc agaattggcg cacgcagtgc cgatccgttc actaatcgaa tggatctgtc
     4801 tctgtctctc tctccacctt cttcttctat tccttcgggc ctgtcgggtc ccctcggggt
     4861 tgggaggtgg gtctgaaacg ataatggtga atatccctgc ctaactctat tcactataga
     4921 aagtacagca aaaactattc ttaaacctac caagcctcct actatcatta tgaataattt
     4981 tatataccac agccaatttg ttatgttaaa ccaattccac aaacttgccc atttatctaa
     5041 ttccaataat tcttgttcat tcttttcttg ctggttttgc gattcttcaa ttaaggagtg
     5101 tattaagctt gtgtaattgt taatttctct gtcccactcc atccaggtcg tgtgattcca
     5161 aatctgttcc agagatttat tactccaact agcattccaa ggcacagcag tggtgcaaat
     5221 gagttttcca gagcaacccc aaatccccag gagctgttga tcctttaggt atctttccac
     5281 agccaggatt cttgcctgga gctgcttgat gccccagact gtgagttgca acagatgctg
     5341 ttgcgcctca atagccctca gcaaattgtt ctgctgctgc actataccag acaataattg
     5401 tctggcctgt accgtcagcg tcattgacgc tgcgcccata gtgcttcctg ctgctcccaa
     5461 gaacccaagg aacaaagctc ctattcccac tgctcttttt tctctctgca ccactcttct
     5521 ctttgccttg gtgggtgcta ctcctaatgg ttcaattttt actactttat atttatataa
     5581 ttcacttctc caattgtccc tcatatctcc tcctccaggt ctgaagatca gcggccgctt
     5641 gctgtgcggt ggtcttactt ttgttttgct cttcctctat cttgtctaaa gcttccttgg
     5701 tgtcttttat ctctatcctt tgatgcacac aatagagggt tgctactgta ttatataatg
     5761 atctaagttc ttctgatcct gtctgaaggg atggttgtag ctgtcccagt atttgtctac
     5821 agccttctga tgtttctaac aggccaggat taactgcgaa tcgttctagc tccctgcttg
     5881 cccatactat atgttttaat ttatattttt tctttccccc tggccttaac cgaatttttt
     5941 cccatcgcga tctaattctc ccccgcttaa tactgacgct ctcgcaccca tctctctcct
     6001 tctagcctcc gctagtcaaa atttttggcg tactcaccag tcgccgcccc tcgcctcttg
     6061 ccgtgcgcgc ttcagcaagc cgagtcctgc gtcgagagag ctcctctggt ttccctttcg
     6121 ctttcaagtc cctgttcggg cgccactgct agagattttc cacactgact aaaagggtct
     6181 gagggatctc tagttaccag agtcacacaa cagacgggca cacactactt gaagcactca
     6241 aggcaaactt tattgaggct taagcagtgg gttccctagt tagccagaga gctcccaggc
     6301 tcagatctgg tctaaccaga gaaacccagt acaggcaaaa cgcgctgctt atatagacct
     6361 cccaccgtac acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg
     6421 gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac
     6481 ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca
     6541 tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata
     6601 aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg
     6661 gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact
     6721 ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta
     6781 ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt
     6841 atgtaacgag atcttggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     6901 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     6961 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     7021 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     7081 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     7141 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     7201 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     7261 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     7321 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     7381 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     7441 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     7501 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     7561 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     7621 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     7681 gtttattgct gataaatctg gagccggtga gcgtgggagc cgcggtatca ttgcagcact
     7741 ggggccagat ggtaagccct cccgtatcgt
//
