LOCUS       sequence-246164-        8251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry plasmid for two-step guide-barcode cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8251)
  AUTHORS   Feldman D, Singh A, Schmid-Burgk JL, Carlson RJ, Mezger A, Garrity 
            AJ, Zhang F, Blainey PC
  TITLE     Optical Pooled Screens in Human Cells.
  JOURNAL   Cell. 2019 Oct 17;179(3):787-799.e17. doi: 
            10.1016/j.cell.2019.09.016.
  PUBMED    31626775
REFERENCE   2  (bases 1 to 8251)
  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"; doi: 
            "10.1016/j.cell.2019.09"; journalName: "Cell"; date: "2019-10-17- 
            17"; volume: "179"; issue: "3"; pages: "787-799.e17"
COMMENT     Sequence Label: LentiGuide-BC-start
FEATURES             Location/Qualifiers
     source          1..8251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..181
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    228..353
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    846..1079
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1264..1308
                     /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    1606..1723
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1773..2021
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1773..1793
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1944..1963
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2109..2697
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2162..2182)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2580..2591)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2700..2716)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2701..2717)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             3222..3402
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(3428..3445)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3434..3658
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3704..4132
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3791..3810)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4001..4022
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4141..4161)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4146..4475
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4326..4461
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4388..4407
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4523..4570
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4589..4963
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    5093..5214
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5130..5149)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5184..5203
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5263..5279)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5263..5279)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5276..5298)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5287..5303
                     /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)."
     promoter        complement(join(5311..5317,5318..5335,5336..5341))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5356..5377
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5494..5511)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5665..6253)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5745..5764)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6424..7215,7216..7284))
                     /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     7047..7066
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7285..7389)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7464..7483)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7655..8034
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8036..8234
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     8189..8209
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     1501 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
     1561 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
     1621 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     1681 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     1741 cagcagagat ccagtttggt taattagcta gcgagggcct atttcccatg attccttcat
     1801 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     1861 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     1921 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     1981 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggagacggg atactgcagt
     2041 cgtctcgact ggctattcat tcgccctttg ggtaagcaca cgtcgaattc gatatcaagc
     2101 ttatcggtaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     2161 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     2221 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     2281 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     2341 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     2401 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     2461 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     2521 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     2581 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     2641 cgcgtcatcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     2701 gataccgtcg acctcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag
     2761 ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag
     2821 tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact
     2881 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgatga caagatatcc
     2941 ttgatctgtg gatctaccac acacaaggct acttccctga ttggcagaac tacacaccag
     3001 ggccagggat cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc
     3061 aagagaaggt agaagaagcc aatgaaggag agaacacccg cttgttacac cctgtgagcc
     3121 tgcatgggat ggatgacccg gagagagaag tattagagtg gaggtttgac agccgcctag
     3181 catttcatca catggcccga gagctgcatc cggactgtac tgggtctctc tggttagacc
     3241 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     3301 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     3361 gatccctcag acccttttag tcagtgtgga aaatctctag cagggcccgt ttaaacccgc
     3421 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3481 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3541 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3601 aagggggagg attgggatga caatagcagg catgctgggg atgcggtggg ctctatggct
     3661 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     3721 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3781 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3841 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3901 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3961 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4021 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     4081 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     4141 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4201 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     4261 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     4321 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4381 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     4441 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     4501 ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt
     4561 ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct
     4621 caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg
     4681 ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag
     4741 cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct
     4801 ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg
     4861 gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc
     4921 ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt
     4981 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     5041 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     5101 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5161 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     5221 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5281 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5341 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5401 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5461 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5521 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5581 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5641 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5701 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5761 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5821 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5881 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5941 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6001 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6061 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6121 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6181 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6241 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6301 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6361 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6421 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6481 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6541 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6601 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6661 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6721 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6781 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6841 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6901 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6961 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7021 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7081 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7141 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7201 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7261 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7321 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7381 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc
     7441 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     7501 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
     7561 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
     7621 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
     7681 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     7741 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     7801 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     7861 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     7921 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     7981 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     8041 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     8101 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     8161 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     8221 gtctatataa gcagcgcgtt ttgcctgtac t
//
