LOCUS       sequence-160503-        7614 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector expressing RAP2A-p2A-E2Crimson.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7614)
  AUTHORS   Park RJ, Wang T, Koundakjian D, Hultquist JF, Lamothe-Molina P, 
            Monel B, Schumann K, Yu H, Krupzcak KM, Garcia-Beltran W, 
            Piechocka-Trocha A, Krogan NJ, Marson A, Sabatini DM, Lander ES, 
            Hacohen N, Walker BD
  TITLE     A genome-wide CRISPR screen identifies a restricted set of HIV host 
            dependency factors.
  JOURNAL   Nat Genet. 2016 Dec 19. doi: 10.1038/ng.3741.
  PUBMED    27992415
REFERENCE   2  (bases 1 to 7614)
  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: "Nat Genet. 
            2016 Dec 19. doi: 10.1038/ng.3741."
COMMENT     Sequence Label: RAP2A-p2A-E2Crimson
FEATURES             Location/Qualifiers
     source          1..7614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..212
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             821..877
                     /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             881..1552
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2009)"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="STENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAKLQ
                     VTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGVVT
                     VTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHMAL
                     KLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHHLF
                     L"
     primer_bind     1273..1292
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1610..1643)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1699..2287
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1752..1772)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2170..2181)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2290..2306)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             2816..2996
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(3058..3646)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3138..3157)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3817..4608,4609..4677))
                     /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     4440..4459
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4678..4782)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4857..4876)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5048..5427
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5429..5627
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5582..5602
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5645..5825
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5872..5997
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6494..6727
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6912..6956
                     /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    7254..7371
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     7429..7445
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(7487..7520)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
       61 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      121 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      181 tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcgggtta attaagccac
      241 catgcgcgag tacaaagtgg tggtgctggg ctcgggcggg gtaggcaaat ccgccctgac
      301 cgtgcagttc gtgaccggca ccttcatcga gaaatacgac cccaccatcg aggacttcta
      361 ccgcaaggag atcgaggtgg attcgtcgcc gtcggtgctg gagatcctgg acacggcggg
      421 caccgagcag ttcgcgtcca tgcgggacct gtacatcaag aacggccagg gcttcatcct
      481 cgtctacagc ctcgtcaacc agcagagctt ccaggacatc aagcccatgc gggaccagat
      541 catccgcgtg aagcggtatg agaaagtgcc agtcatcttg gttgggaaca aagtggacct
      601 ggaaagtgag agagaagtat cgtccagcga aggcagagcc cttgctgaag agtggggctg
      661 cccctttatg gaaacttccg ctaagagtaa aacaatggtg gacgaactct ttgcagaaat
      721 tgtgaggcag atgaactatg ctgctcagcc tgacaaagat gacccatgct gttctgcatg
      781 taacatacaa gcggccgcag cggccgcagg aggtggagga gctacaaatt tcagcctgct
      841 gaagcaggcc ggcgacgtgg aggagaaccc cggccccaat agcactgaga acgtcatcaa
      901 gcccttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat
      961 cgagggcgtg ggcgagggca agccctacga gggcacccag accgccaagc tgcaagtgac
     1021 caagggcggc cccctgccct tcgcctggga catcctgtcc ccccagttct tctacggctc
     1081 caaggcgtac atcaagcacc ccgccgacat ccccgactac ctcaagcagt ccttccccga
     1141 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
     1201 ggactcctcc ctgcaggacg gcaccctcat ctaccacgtg aagttcatcg gcgtgaactt
     1261 cccctccgac ggccccgtaa tgcagaagaa gactctgggc tgggagccct ccactgagcg
     1321 caactacccc cgcgacggcg tgctgaaggg cgagaaccac atggcgctga agctgaaggg
     1381 cggcggccac tacctgtgtg agttcaagtc catctacatg gccaagaagc ccgtgaagct
     1441 gcccggctac cactacgtgg actacaagct cgacatcacc tcccacaacg aggactacac
     1501 cgtggtggag cagtacgagc gcgccgaggc ccgccaccac ctgttcctgt gatgtacaag
     1561 taaagcggcc ggccgccagc acagtggtcg aaattcgtcg agggacctaa taacttcgta
     1621 tagcatacat tatacgaagt tatacatgtt taagggttcc ggttccacta ggtacaattc
     1681 gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     1741 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     1801 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     1861 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     1921 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     1981 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     2041 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     2101 tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     2161 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     2221 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     2281 tccccgcatc gataccgtcg acctcgatcg agacctagaa aaacatggag caatcacaag
     2341 tagcaataca gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggagga
     2401 ggtgggtttt ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgt
     2461 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg
     2521 aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattggca
     2581 gaactacaca ccagggccag ggatcagata tccactgacc tttggatggt gctacaagct
     2641 agtaccagtt gagcaagaga aggtagaaga agccaatgaa ggagagaaca cccgcttgtt
     2701 acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtattag agtggaggtt
     2761 tgacagccgc ctagcatttc atcacatggc ccgagagctg catccggact gtactgggtc
     2821 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     2881 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     2941 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagcat
     3001 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3061 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3121 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3181 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3241 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3301 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3361 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3421 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3481 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3541 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3601 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3661 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3721 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3781 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3841 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     3901 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     3961 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4021 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4081 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4141 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4201 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4261 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4321 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4381 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4441 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4501 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4561 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4621 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4681 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4741 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     4801 gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca
     4861 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
     4921 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
     4981 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
     5041 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
     5101 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     5161 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     5221 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     5281 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     5341 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     5401 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
     5461 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     5521 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
     5581 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg
     5641 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     5701 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     5761 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     5821 tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga
     5881 cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt
     5941 acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt
     6001 attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag
     6061 aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt
     6121 aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca
     6181 tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat
     6241 tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa
     6301 gagcaaaaca aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg
     6361 gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa
     6421 ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa
     6481 gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg
     6541 gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc
     6601 agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct
     6661 ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac
     6721 agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga
     6781 atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt
     6841 gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa
     6901 accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga
     6961 attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag
     7021 gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg
     7081 gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg
     7141 aaggaataga agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg
     7201 gatcggcact gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa
     7261 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca
     7321 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat
     7381 tacagggaca gcagagatcc agtttggtta gtaccgggcc cgctctatcg aggtcgacgg
     7441 tatcgataag ctcgcttcac gagattccag caggtcgagg gacctaataa cttcgtatag
     7501 catacattat acgaagttat attaagggtt ccaagcttaa gcggccggcc gctgaaagac
     7561 cccacctgta ggtttggcaa gctagcggct cgagtggctc cggtgcccgt cagt
//
