LOCUS       sequence-192144-        7652 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hgRNA-A51 in a lentiviral backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7652)
  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 7652)
  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-A51_pLKO-Hyg
FEATURES             Location/Qualifiers
     source          1..7652
                     /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    2257..2374
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2423..2933
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2852..2870
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3023..4048
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3023..4048
                     /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(4072..4088)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4073..4089)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4171..4404
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4476..4597
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4513..4532)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4567..4586
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4637..4772
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4792..4811)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4793..4811)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4821..4838)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4821..4837)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4830..4852)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4979..5434
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5066..5085)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5276..5297
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5460..5564
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5565..5633,5634..6425)
                     /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(5783..5802)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6596..7184
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7085..7104
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7338..7355
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7472..7493
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7508..7513,7514..7531,7532..7538)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7546..7562
                     /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     7551..7573
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7570..7586
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7570..7586
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7605..7625
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7607..7625
                     /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 cggtagacgc ggtcacactg
     2101 atgcagctag tatgcacctc caccccacag tggggttaga gctagaaata gcaagttaac
     2161 ctaaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttgaatt
     2221 ctcgacctcg agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg
     2281 ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga
     2341 attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga
     2401 tccactttgg ccgcggctcg agggggttgg ggttgcgcct tttccaaggc agccctgggt
     2461 ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa acgcagcggc gccgaccctg
     2521 ggtctcgcac attcttcacg tccgttcgca gcgtcacccg gatcttcgcc gctacccttg
     2581 tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg
     2641 cggcgtgccg gacgtgacaa acggaagccg cacgtctcac tagtaccctc gcagacggac
     2701 agcgccaggg agcaatggca gcgcgccgac cgcgatgggc tgtggccaat agcggctgct
     2761 cagcagggcg cgccgagagc agcggccggg aaggggcggt gcgggaggcg gggtgtgggg
     2821 cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag cctccggagc
     2881 gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga cctctctccc cagggggatc
     2941 caccggatca agcttgccag ctggggcgcc ctctggtaag gttgggaagc cctgcagatc
     3001 tgatcccggg gggcaatgag atatgaaaaa gcctgaactc accgcgacgt ctgtcgagaa
     3061 gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga
     3121 atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg
     3181 cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc
     3241 gattccggaa gtgcttgaca ttggggaatt tagcgagagc ctgacctatt gcatctcccg
     3301 ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca
     3361 gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt
     3421 cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc
     3481 gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc
     3541 cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca
     3601 cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc
     3661 ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt
     3721 cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca
     3781 tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca
     3841 actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg
     3901 cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag
     3961 cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc
     4021 cagcactcgt ccgagggcaa aggaatagag tagatgccga ccgggatcta tcgataccgt
     4081 cgacctcgag ggggggcccg gtacctttaa gaccaatgac ttacaaggca gctgtagatc
     4141 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     4201 aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc
     4261 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     4321 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     4381 agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat
     4441 aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg
     4501 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4561 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc
     4621 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     4681 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     4741 ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt
     4801 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     4861 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     4921 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     4981 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     5041 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     5101 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     5161 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     5221 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     5281 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     5341 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     5401 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     5461 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5521 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5581 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5641 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5701 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5761 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5821 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5881 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5941 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6001 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     6061 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     6121 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     6181 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     6241 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     6301 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6361 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6421 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6481 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6541 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6601 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6661 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6721 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6781 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6841 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6901 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6961 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7021 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     7081 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     7141 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7201 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     7261 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     7321 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     7381 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     7441 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     7501 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     7561 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
     7621 aaagggaaca aaagctggag ctgcaagctt aa
//
