LOCUS       sequence-243214-        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"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2607..2855
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2607..2627
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2778..2797
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2943..3531
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2996..3016)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3414..3425)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3534..3550)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3535..3551)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4056..4236
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4262..4279)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4268..4492
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4538..4966
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4625..4644)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4835..4856
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4975..4995)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4980..5309
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5160..5295
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5222..5241
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5357..5404
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5423..5797
                     /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    5927..6048
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5964..5983)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6018..6037
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6097..6113)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6097..6113)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6110..6132)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6121..6137
                     /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(6145..6151,6152..6169,6170..6175))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6190..6211
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6328..6345)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6499..7087)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6579..6598)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7258..8049,8050..8118))
                     /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     7881..7900
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8119..8223)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta gctagcgagg gcctatttcc catgattcct tcatatttgc
     2641 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     2701 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     2761 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     2821 tggctttata tatcttgtgg aaaggacgaa acaccggaga cgggatactg cagtcgtctc
     2881 gactggctat tcattcgccc tttgggtaag cacacgtcga attcgatatc aagcttatcg
     2941 gtaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     3001 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     3061 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     3121 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     3181 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     3241 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     3301 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     3361 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     3421 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     3481 atcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
     3541 gtcgacctcg agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca
     3601 atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac
     3661 ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     3721 aagaaaaggg gggactggaa gggctaattc actcccaacg atgacaagat atccttgatc
     3781 tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag
     3841 ggatcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga
     3901 aggtagaaga agccaatgaa ggagagaaca cccgcttgtt acaccctgtg agcctgcatg
     3961 ggatggatga cccggagaga gaagtattag agtggaggtt tgacagccgc ctagcatttc
     4021 atcacatggc ccgagagctg catccggact gtactgggtc tctctggtta gaccagatct
     4081 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     4141 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     4201 tcagaccctt ttagtcagtg tggaaaatct ctagcagggc ccgtttaaac ccgctgatca
     4261 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     4321 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     4381 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     4441 gaggattggg atgacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     4501 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     4561 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     4621 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4681 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4741 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4801 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4861 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     4921 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     4981 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     5041 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     5101 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     5161 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     5221 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     5281 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     5341 gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata
     5401 cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc
     5461 gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt
     5521 cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt
     5581 ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga
     5641 gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc
     5701 catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg
     5761 caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtgctacgag atttcgattc
     5821 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     5881 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     5941 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     6001 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     6061 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     6121 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     6181 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     6241 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6301 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     6361 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     6421 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     6481 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     6541 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     6601 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     6661 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6721 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6781 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6841 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6901 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6961 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     7021 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     7081 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     7141 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     7201 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     7261 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     7321 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     7381 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     7441 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     7501 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     7561 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     7621 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     7681 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7741 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7801 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7861 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7921 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7981 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     8041 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     8101 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     8161 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     8221 gcgcacattt ccccgaaaag tgccacctga c
//
