LOCUS       sequence-160504-        8805 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector expressing ALCAM-p2A-E2Crimson.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8805)
  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 8805)
  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: ALCAM-p2A-E2Crimson
FEATURES             Location/Qualifiers
     source          1..8805
                     /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"
     regulatory      236..245
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2012..2068
                     /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             2072..2743
                     /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     2464..2483
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2801..2834)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2890..3478
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2943..2963)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3361..3372)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3481..3497)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             4007..4187
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4249..4837)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4329..4348)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5008..5799,5800..5868))
                     /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     5631..5650
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5869..5973)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6048..6067)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6239..6618
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6620..6818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6773..6793
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             6836..7016
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7063..7188
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7685..7918
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8103..8147
                     /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    8445..8562
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     8620..8636
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(8678..8711)
                     /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 catggagtca aaaggtgcct cttcctgcag gctgctgttc tgtttgctga tttccgcaac
      301 agtgttccgc cccggactcg gatggtatac cgtgaattct gcttatggtg atacgattat
      361 cattccatgc cggttggatg ttccacagaa tcttatgttc ggaaaatgga agtacgagaa
      421 gcccgatggc tctcctgttt ttattgcctt ccggtccagc acaaaaaaga gcgtgcagta
      481 cgacgacgtg cctgagtaca aggacaggct taacctgtcc gagaactaca ctctgtccat
      541 ctcaaatgcc cggatttccg atgagaagcg gttcgtatgc atgctggtaa ccgaagataa
      601 tgtattcgag gcgcccacca tcgtgaaagt gtttaaacaa ccgtcaaagc ccgagatcgt
      661 ttcaaaagca ctcttcttgg agactgaaca actcaagaag ttgggggatt gcatcagtga
      721 ggacagctat ccggacggaa acatcacttg gtatcggaac ggcaaagttc tccacccgct
      781 ggagggcgct gtggtcatca tcttcaagaa ggagatggac cctgttacgc agctttacac
      841 aatgaccagc accttggagt acaaaactac taaagcagat atccagatgc cttttacatg
      901 ctccgtcact tactacggac cttcaggaca gaagaccatc cattctgagc aggcggtatt
      961 cgatatttac taccccacag aacaggtaac tatacaggtg ctccccccta aaaacgccat
     1021 caaagaggga gacaacatca cccttaagtg tcttggaaat ggtaatcctc cgccagaaga
     1081 gtttctgttt tatcttcctg gacagcctga gggcatcaga agcagcaata cgtataccct
     1141 gacagatgtc cggagaaacg ctacaggcga ttacaagtgc tcactgatcg acaaaaagag
     1201 catgattgct tccaccgcaa tcaccgtgca ctatctggac ctgtctttga atccatcagg
     1261 ggaagtcacc aggcagatcg gagacgccct tcctgtgtca tgtacaattt ctgcctctag
     1321 aaatgcgaca gtagtctgga tgaaggacaa tatcaggctc agaagcagtc cttcctttag
     1381 tagtctccac taccaggacg ccgggaacta cgtttgcgag accgctctgc aggaggttga
     1441 gggccttaaa aaaagagaga gtttgacatt gatcgtggag gggaaacccc agatcaagat
     1501 gaccaaaaaa actgatccca gcggtctctc taaaacaatc atctgccacg tggaggggtt
     1561 ccctaagccg gccatccagt ggacaatcac aggcagcggc agtgtcatta atcagaccga
     1621 ggaatctccg tacattaatg gcagatacta ctctaaaatc atcatttcac cggaggaaaa
     1681 cgtcacgctg acctgtacag cagagaacca gttggagcgc acagttaatt cactgaacgt
     1741 ttccgctatc agtattcccg agcatgacga ggccgacgag atcagcgatg agaaccgcga
     1801 aaaggtgaat gatcaagcta agctcatagt gggcatagtg gtggggttgc tgctggcggc
     1861 tctggttgca ggcgttgtgt actggctgta tatgaaaaag agcaaaaccg ctagcaagca
     1921 tgtcaataag gaccttggaa acatggagga aaataagaag ctggaagaga acaaccacaa
     1981 gacagaggcc gcggccgcag gaggtggagg agctacaaat ttcagcctgc tgaagcaggc
     2041 cggcgacgtg gaggagaacc ccggccccaa tagcactgag aacgtcatca agcccttcat
     2101 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcgt
     2161 gggcgagggc aagccctacg agggcaccca gaccgccaag ctgcaagtga ccaagggcgg
     2221 ccccctgccc ttcgcctggg acatcctgtc cccccagttc ttctacggct ccaaggcgta
     2281 catcaagcac cccgccgaca tccccgacta cctcaagcag tccttccccg agggcttcaa
     2341 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     2401 cctgcaggac ggcaccctca tctaccacgt gaagttcatc ggcgtgaact tcccctccga
     2461 cggccccgta atgcagaaga agactctggg ctgggagccc tccactgagc gcaactaccc
     2521 ccgcgacggc gtgctgaagg gcgagaacca catggcgctg aagctgaagg gcggcggcca
     2581 ctacctgtgt gagttcaagt ccatctacat ggccaagaag cccgtgaagc tgcccggcta
     2641 ccactacgtg gactacaagc tcgacatcac ctcccacaac gaggactaca ccgtggtgga
     2701 gcagtacgag cgcgccgagg cccgccacca cctgttcctg tgatgtacaa gtaaagcggc
     2761 cggccgccag cacagtggtc gaaattcgtc gagggaccta ataacttcgt atagcataca
     2821 ttatacgaag ttatacatgt ttaagggttc cggttccact aggtacaatt cgatatcaag
     2881 cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     2941 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     3001 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     3061 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     3121 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     3181 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     3241 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     3301 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     3361 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     3421 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     3481 cgataccgtc gacctcgatc gagacctaga aaaacatgga gcaatcacaa gtagcaatac
     3541 agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt
     3601 tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag
     3661 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
     3721 tatccttgat ctgtggatct accacacaca aggctacttc cctgattggc agaactacac
     3781 accagggcca gggatcagat atccactgac ctttggatgg tgctacaagc tagtaccagt
     3841 tgagcaagag aaggtagaag aagccaatga aggagagaac acccgcttgt tacaccctgt
     3901 gagcctgcat gggatggatg acccggagag agaagtatta gagtggaggt ttgacagccg
     3961 cctagcattt catcacatgg cccgagagct gcatccggac tgtactgggt ctctctggtt
     4021 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     4081 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     4141 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagca tgtgagcaaa
     4201 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4261 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4321 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4381 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4441 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4501 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4561 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4621 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4681 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4741 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4801 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4861 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4921 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4981 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5041 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5101 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5161 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5221 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5281 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5341 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5401 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5461 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5521 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5581 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5641 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5701 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5761 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5821 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5881 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5941 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6001 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     6061 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     6121 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     6181 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
     6241 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     6301 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     6361 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     6421 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     6481 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     6541 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     6601 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     6661 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     6721 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     6781 ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc
     6841 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     6901 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     6961 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
     7021 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
     7081 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     7141 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     7201 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     7261 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     7321 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     7381 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     7441 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     7501 aaaagtaaga ccaccgcaca gcaagcggcc ggccgctgat cttcagacct ggaggaggag
     7561 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     7621 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     7681 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     7741 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     7801 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     7861 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     7921 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     7981 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     8041 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     8101 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     8161 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     8221 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     8281 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
     8341 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac
     8401 tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     8461 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     8521 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     8581 agcagagatc cagtttggtt agtaccgggc ccgctctatc gaggtcgacg gtatcgataa
     8641 gctcgcttca cgagattcca gcaggtcgag ggacctaata acttcgtata gcatacatta
     8701 tacgaagtta tattaagggt tccaagctta agcggccggc cgctgaaaga ccccacctgt
     8761 aggtttggca agctagcggc tcgagtggct ccggtgcccg tcagt
//
