LOCUS       sequence-163149-        7290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector expressing an sgRNA targeting ALCAM. NOTE: This 
            plasmid does NOT contain Cas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7290)
  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 7290)
  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: pLenti-sgALCAM
FEATURES             Location/Qualifiers
     source          1..7290
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..231
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             232..412
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    459..584
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1077..1310
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1495..1539
                     /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"
     promoter        1717..1965
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1717..1737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1888..1907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1986..2061
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2109..2226
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2295..2506
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2532..3131
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2532..2551)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3028..3048
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3147..3735
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3200..3220)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3618..3629)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3807..4040
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4118..4239
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4155..4174)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4209..4228
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4279..4414
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4434..4453)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4435..4453)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4463..4480)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4463..4479)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4472..4494)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4621..5076
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4708..4727)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4918..4939
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5102..5206
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5207..5275,5276..6067)
                     /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(5425..5444)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6238..6826
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6727..6746
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6980..6997
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7114..7135
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7150..7155,7156..7173,7174..7180)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7188..7204
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7193..7215
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7212..7228
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7212..7228
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7247..7267
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7249..7267
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
       61 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      121 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      181 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      241 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      301 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      361 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      421 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      481 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      541 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      601 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
      661 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
      721 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
      781 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
      841 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
      901 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
      961 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1021 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1081 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1141 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1201 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1261 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1321 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1381 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1441 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1501 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1561 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1621 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     1681 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct
     1741 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta
     1801 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1861 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1921 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgtgct acatcccctt
     1981 gaagggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     2041 aaagtggcac cgagtcggtg cttttttgaa ttctagatct tgagacaaat ggcagtattc
     2101 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     2161 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     2221 aattttcggg tttattacag ggacagcaga gatccacttt ggcgccggct cgagtggctc
     2281 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     2341 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     2401 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     2461 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtgt cgtgacgcgg
     2521 gatccgccac catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     2581 ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     2641 tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     2701 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     2761 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     2821 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     2881 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac caccagggca
     2941 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     3001 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     3061 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     3121 ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat
     3181 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     3241 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     3301 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     3361 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     3421 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     3481 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     3541 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     3601 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     3661 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     3721 gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg tagatcttag
     3781 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
     3841 tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     3901 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     3961 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     4021 agtgtggaaa atctctagca gtacgtatag tagttcatgt catcttatta ttcagtattt
     4081 ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa
     4141 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4201 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc
     4261 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4321 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4381 ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata
     4441 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     4501 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     4561 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg
     4621 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4681 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4741 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4801 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     4861 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4921 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     4981 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5041 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     5101 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5161 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5221 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5281 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5341 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5401 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5461 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5521 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5581 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5641 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5701 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5761 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5821 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5881 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5941 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6001 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6061 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6121 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6181 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6241 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6301 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6361 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6421 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6481 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6541 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6601 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6661 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6721 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6781 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6841 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     6901 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     6961 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     7021 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     7081 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     7141 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     7201 acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca
     7261 ctaaagggaa caaaagctgg agctgcaagc
//
