LOCUS       sequence-192142-        7627 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  hgRNA-A26 in a lentiviral backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7627)
  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 7627)
  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-A26_pLKO-Hyg
FEATURES             Location/Qualifiers
     source          1..7627
                     /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    2232..2349
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2398..2908
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2827..2845
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2998..4023
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2998..4023
                     /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(4047..4063)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4048..4064)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4146..4379
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4451..4572
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4488..4507)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4542..4561
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4612..4747
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4767..4786)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4768..4786)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4796..4813)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4796..4812)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4805..4827)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4954..5409
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5041..5060)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5251..5272
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5435..5539
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5540..5608,5609..6400)
                     /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(5758..5777)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6571..7159
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7060..7079
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7313..7330
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7447..7468
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7483..7488,7489..7506,7507..7513)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7521..7537
                     /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     7526..7548
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7545..7561
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7545..7561
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7580..7600
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7582..7600
                     /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 acctccaccc
     2101 cacagtgggg ttagagctag aaatagcaag ttaacctaag gctagtccgt tatcaacttg
     2161 aaaaagtggc accgagtcgg tgcttttttt gaattctcga cctcgagaca aatggcagta
     2221 ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata
     2281 gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt
     2341 caaaattttc gggtttatta cagggacagc agagatccac tttggccgcg gctcgagggg
     2401 gttggggttg cgccttttcc aaggcagccc tgggtttgcg cagggacgcg gctgctctgg
     2461 gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct tcacgtccgt
     2521 tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccggcga cgcttcctgc
     2581 tccgccccta agtcgggaag gttccttgcg gttcgcggcg tgccggacgt gacaaacgga
     2641 agccgcacgt ctcactagta ccctcgcaga cggacagcgc cagggagcaa tggcagcgcg
     2701 ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg agagcagcgg
     2761 ccgggaaggg gcggtgcggg aggcggggtg tggggcggta gtgtgggccc tgttcctgcc
     2821 cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg gctccctcgt
     2881 tgaccgaatc accgacctct ctccccaggg ggatccaccg gatcaagctt gccagctggg
     2941 gcgccctctg gtaaggttgg gaagccctgc agatctgatc ccggggggca atgagatatg
     3001 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc
     3061 gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
     3121 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt
     3181 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg
     3241 gaatttagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa
     3301 gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg
     3361 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc
     3421 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac
     3481 tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg
     3541 atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc
     3601 aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg
     3661 ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt
     3721 atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg
     3781 ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc
     3841 aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc
     3901 gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt
     3961 gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa
     4021 tagagtagat gccgaccggg atctatcgat accgtcgacc tcgagggggg gcccggtacc
     4081 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     4141 gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg
     4201 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     4261 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     4321 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     4381 tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga
     4441 gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4501 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4561 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc
     4621 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4681 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     4741 ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     4801 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     4861 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     4921 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     4981 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     5041 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     5101 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     5161 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     5221 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     5281 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     5341 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     5401 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     5461 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     5521 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     5581 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     5641 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     5701 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     5761 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5821 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     5881 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5941 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     6001 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     6061 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     6121 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     6181 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     6241 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6301 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6361 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6421 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6481 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6541 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6601 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6661 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6721 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6781 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6841 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6901 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6961 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     7021 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     7081 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     7141 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     7201 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     7261 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     7321 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     7381 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     7441 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     7501 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     7561 ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca
     7621 agcttaa
//
