LOCUS       sequence-365635-        5687 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Tier-3 vector for stable integration of up to three cassettes via 
            lentiviral transduction in inverted direction with polyA 
            (PCMV-5'LTR-Psi-gag-env-A3-pA::A2-pA::A1-pA-WPRE-3'LTR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5687)
  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 5687)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pTS1108_Tier3(Lenti_inverse)
FEATURES             Location/Qualifiers
     source          1..5687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     411..430
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     LTR             573..806
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    878..1466
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             984..995
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1393..1413
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(1640..1657)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1646..1870
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2016..2032)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2016..2032)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    2232..2349
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(2647..2691)
                     /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    2876..3109
                     /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    3602..3727
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             3774..3954
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(3972..4170)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3997..4017)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4172..4475
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4523..4627
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4628..4696,4697..5488)
                     /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(4846..4865)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5609..5687,1..510)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
       61 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
      121 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
      181 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
      241 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
      301 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
      361 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
      421 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
      481 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
      541 ctggcctttt gctggccttt tgctcacatg tctgctagag attttccaca ctgactaaaa
      601 gggtctgagg gatctctagt taccagagtc acacaacaga cgggcacaca ctacttgaag
      661 cactcaaggc aaactttatt gaggcttaag cagtgggttc cctagttagc cagagagctc
      721 ccaggctcag atctggtcta accagagaaa cccagtacaa gcaaaaagca gatcttgtct
      781 tcgttgggag tgaattagcc cttccagtcc ccccttttct tttaaaaagt ggctaagatc
      841 tacagctgcc ttgtaagtca ttggtcttaa agtcgacgcg gggaggcggc ccaaagggag
      901 atccgactcg tctgagggcg aaggcgaaga cgcggaagag gccgcagagc cggcagcagg
      961 ccgcgggaag gaaggtccgc tggattgagg gccgaaggga cgtagcagaa ggacgtcccg
     1021 cgcagaatcc aggtggcaac acaggcgagc agccaaggaa aggacgatga tttccccgac
     1081 aacaccacgg aattgtcagt gcccaacagc cgagcccctg tccagcagcg ggcaaggcag
     1141 gcggcgatga gttccgccgt ggcaataggg agggggaaag cgaaagtccc ggaaaggagc
     1201 tgacaggtgg tggcaatgcc ccaaccagtg ggggttgcgt cagcaaacac agtgcacacc
     1261 acgccacgtt gcctgacaac gggccacaac tcctcataaa gagacagcaa ccaggattta
     1321 tacaaggagg agaaaatgaa agccatacgg gaagcaatag catgatacaa aggcattaaa
     1381 gcagcgtatc cacatagcgt aaaaggagca acatagttaa gaataccagt caatctttca
     1441 caaattttgt aatccagagg ttgattgatc tggcacgaca ggtttcccga ctggaaagcg
     1501 ggcagtgagc gcaacgcaat taatagctac gcgtggtacc ctcgaggagc tcttcgaagc
     1561 ggaattcacc atgactagtg ctagcggatc caccggtgtc tagaaagctt tgaggccggc
     1621 ctgcaggccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     1681 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     1741 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     1801 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     1861 ggctctatgg tagcttagga ggcggaaaga accagcgcgg gagaccgtca gttggtctcc
     1921 agctatacag gtgcaccgct ttataataaa actgatacag agaacgttat catgtgtgtt
     1981 ggtttttttt gtgtgtgcgg tttaagggag cctcggtcat agctgtttcc tgtgtaaaac
     2041 gacggccagc gcgggagacg gtcagttcgt ctccagctca gaaattatga gagcgtttaa
     2101 taaagtattg actatggttg acaaaacgtg tgttggagtt ttttgtgtgc tttgtcatca
     2161 actgagccag tagcgcgtga ccaagtatcc ccgcttcttc tcgcgtcact gctgtccctg
     2221 taataaaccc gaaaattttg aatttttgta atttgttttt gtaattcttt agtttgtatg
     2281 tctgttgcta ttatgtctac tattctttcc cctgcactgt accccccaat cccccctttt
     2341 cttttaaaat tgtggatgaa tactgccatt tgtctgcaga attggcgcac gcagtgccga
     2401 tccgttcact aatcgaatgg atctgtctct gtctctctct ccaccttctt cttctattcc
     2461 ttcgggcctg tcgggtcccc tcggggttgg gaggtgggtc tgaaacgata atggtgaata
     2521 tccctgccta actctattca ctatagaaag tacagcaaaa actattctta aacctaccaa
     2581 gcctcctact atcattatga ataattttat ataccacagc caatttgtta tgttaaacca
     2641 attccacaaa cttgcccatt tatctaattc caataattct tgttcattct tttcttgctg
     2701 gttttgcgat tcttcaatta aggagtgtat taagcttgtg taattgttaa tttctctgtc
     2761 ccactccatc caggtcgtgt gattccaaat ctgttccaga gatttattac tccaactagc
     2821 attccaaggc acagcagtgg tgcaaatgag ttttccagag caaccccaaa tccccaggag
     2881 ctgttgatcc tttaggtatc tttccacagc caggattctt gcctggagct gcttgatgcc
     2941 ccagactgtg agttgcaaca gatgctgttg cgcctcaata gccctcagca aattgttctg
     3001 ctgctgcact ataccagaca ataattgtct ggcctgtacc gtcagcgtca ttgacgctgc
     3061 gcccatagtg cttcctgctg ctcccaagaa cccaaggaac aaagctccta ttcccactgc
     3121 tcttttttct ctctgcacca ctcttctctt tgccttggtg ggtgctactc ctaatggttc
     3181 aatttttact actttatatt tatataattc acttctccaa ttgtccctca tatctcctcc
     3241 tccaggtctg aagatcagcg gccgcttgct gtgcggtggt cttacttttg ttttgctctt
     3301 cctctatctt gtctaaagct tccttggtgt cttttatctc tatcctttga tgcacacaat
     3361 agagggttgc tactgtatta tataatgatc taagttcttc tgatcctgtc tgaagggatg
     3421 gttgtagctg tcccagtatt tgtctacagc cttctgatgt ttctaacagg ccaggattaa
     3481 ctgcgaatcg ttctagctcc ctgcttgccc atactatatg ttttaattta tattttttct
     3541 ttccccctgg ccttaaccga attttttccc atcgcgatct aattctcccc cgcttaatac
     3601 tgacgctctc gcacccatct ctctccttct agcctccgct agtcaaaatt tttggcgtac
     3661 tcaccagtcg ccgcccctcg cctcttgccg tgcgcgcttc agcaagccga gtcctgcgtc
     3721 gagagagctc ctctggtttc cctttcgctt tcaagtccct gttcgggcgc cactgctaga
     3781 gattttccac actgactaaa agggtctgag ggatctctag ttaccagagt cacacaacag
     3841 acgggcacac actacttgaa gcactcaagg caaactttat tgaggcttaa gcagtgggtt
     3901 ccctagttag ccagagagct cccaggctca gatctggtct aaccagagaa acccagtaca
     3961 ggcaaaacgc gctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
     4021 atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac
     4081 tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac
     4141 gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga
     4201 tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc
     4261 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     4321 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     4381 gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc
     4441 agccaggcgg gccatttacc gtaagttatg taacgagatc ttggtttctt agacgtcagg
     4501 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4561 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4621 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4681 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     4741 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4801 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4861 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4921 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4981 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5041 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5101 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5161 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5221 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5281 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5341 tgggagccgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5401 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5461 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5521 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5581 tccccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5641 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccg
//
