LOCUS       sequence-192137-        7622 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hgRNA-D21 in a lentiviral backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7622)
  AUTHORS   Kalhor R, Mali P, Church GM
  TITLE     Rapidly evolving homing CRISPR barcodes.
  JOURNAL   Nat Methods. 2017 Feb;14(2):195-200. doi: 10.1038/nmeth.4108. Epub 
            2016 Dec 5.
  PUBMED    27918539
REFERENCE   2  (bases 1 to 7622)
  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: 
            "10.1038/nmeth.4108"; journalName: "Nat Methods"; date: "2017-02"; 
            volume: "14"; issue: "2"; pages: "195-200"
COMMENT     Sequence Label: hgRNA-D21_pLKO-Hyg
FEATURES             Location/Qualifiers
     source          1..7622
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /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        1833..2081
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1833..1853
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2004..2023
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2227..2344
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2393..2903
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2822..2840
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2993..4018
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2993..4018
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(4042..4058)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4043..4059)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4141..4374
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4446..4567
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4483..4502)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4537..4556
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4607..4742
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4762..4781)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4763..4781)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4791..4808)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4791..4807)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4800..4822)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4949..5404
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5036..5055)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5246..5267
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5430..5534
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5535..5603,5604..6395)
                     /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(5753..5772)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6566..7154
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7055..7074
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7308..7325
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7442..7463
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7478..7483,7484..7501,7502..7508)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7516..7532
                     /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     7521..7543
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7540..7556
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7540..7556
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7575..7595
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7577..7595
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     1741 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac
     1801 gagactagcc tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat
     1861 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     1921 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     1981 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     2041 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggtcacgct acggccaagg
     2101 tggggttaga gctagaaata gcaagttaac ctaaggctag tccgttatca acttgaaaaa
     2161 gtggcaccga gtcggtgctt tttttgaatt ctcgacctcg agacaaatgg cagtattcat
     2221 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     2281 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     2341 ttttcgggtt tattacaggg acagcagaga tccactttgg ccgcggctcg agggggttgg
     2401 ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg
     2461 gttccgggaa acgcagcggc gccgaccctg ggtctcgcac attcttcacg tccgttcgca
     2521 gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt cctgctccgc
     2581 ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg
     2641 cacgtctcac tagtaccctc gcagacggac agcgccaggg agcaatggca gcgcgccgac
     2701 cgcgatgggc tgtggccaat agcggctgct cagcagggcg cgccgagagc agcggccggg
     2761 aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc
     2821 ggtgttccgc attctgcaag cctccggagc gcacgtcggc agtcggctcc ctcgttgacc
     2881 gaatcaccga cctctctccc cagggggatc caccggatca agcttgccag ctggggcgcc
     2941 ctctggtaag gttgggaagc cctgcagatc tgatcccggg gggcaatgag atatgaaaaa
     3001 gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc
     3061 cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg
     3121 gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt
     3181 ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt
     3241 tagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct
     3301 gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc
     3361 tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca
     3421 atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca
     3481 aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct
     3541 ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa
     3601 tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg
     3661 ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga
     3721 gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg
     3781 ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt
     3841 cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac
     3901 tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga
     3961 agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagag
     4021 tagatgccga ccgggatcta tcgataccgt cgacctcgag ggggggcccg gtacctttaa
     4081 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     4141 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
     4201 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     4261 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     4321 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta
     4381 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
     4441 agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4501 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4561 tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac
     4621 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     4681 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     4741 cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     4801 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     4861 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     4921 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     4981 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     5041 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5101 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5161 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5221 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5281 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     5341 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     5401 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5461 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5521 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5581 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5641 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5701 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5761 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5821 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5881 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5941 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6001 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6061 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6121 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6181 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6241 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6301 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6361 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6421 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6481 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6541 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6601 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6661 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6721 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6781 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6841 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6901 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6961 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     7021 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7081 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7141 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7201 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     7261 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     7321 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     7381 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     7441 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     7501 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     7561 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt
     7621 aa
//
