LOCUS       sequence-192150-        7654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FISSEQ-hgRNA-Design1 in a lentiviral backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7654)
  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 7654)
  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: FISSEQ-hgRNA-Design1
FEATURES             Location/Qualifiers
     source          1..7654
                     /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    2259..2376
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2425..2935
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2854..2872
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3025..4050
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3025..4050
                     /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(4074..4090)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4075..4091)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4173..4406
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4478..4599
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4515..4534)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4569..4588
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4639..4774
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4794..4813)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4795..4813)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4823..4840)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4823..4839)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4832..4854)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4981..5436
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5068..5087)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5278..5299
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5462..5566
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5567..5635,5636..6427)
                     /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(5785..5804)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6598..7186
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7087..7106
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7340..7357
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7474..7495
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7510..7515,7516..7533,7534..7540)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7548..7564
                     /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     7553..7575
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7572..7588
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7572..7588
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7607..7627
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7609..7627
                     /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 cggtggagca attccacaac
     2101 acgggttaga gctagaaata gcaagttaac ctaaggctag tccgttatca acttgaaaaa
     2161 gtggcaccga gtcggtgcca ctgttgtctt ataccaactt tccttttttt cctgcaggaa
     2221 ttctcgacct cgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
     2281 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     2341 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
     2401 gatccacttt ggccgcggct cgagggggtt ggggttgcgc cttttccaag gcagccctgg
     2461 gtttgcgcag ggacgcggct gctctgggcg tggttccggg aaacgcagcg gcgccgaccc
     2521 tgggtctcgc acattcttca cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct
     2581 tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt
     2641 cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc actagtaccc tcgcagacgg
     2701 acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca atagcggctg
     2761 ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg
     2821 ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca agcctccgga
     2881 gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc cccaggggga
     2941 tccaccggat caagcttgcc agctggggcg ccctctggta aggttgggaa gccctgcaga
     3001 tctgatcccg gggggcaatg agatatgaaa aagcctgaac tcaccgcgac gtctgtcgag
     3061 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     3121 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     3181 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     3241 ccgattccgg aagtgcttga cattggggaa tttagcgaga gcctgaccta ttgcatctcc
     3301 cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg
     3361 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     3421 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     3481 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     3541 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     3601 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     3661 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     3721 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     3781 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     3841 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     3901 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     3961 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     4021 cccagcactc gtccgagggc aaaggaatag agtagatgcc gaccgggatc tatcgatacc
     4081 gtcgacctcg agggggggcc cggtaccttt aagaccaatg acttacaagg cagctgtaga
     4141 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag
     4201 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga
     4261 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct
     4321 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt
     4381 ttagtcagtg tggaaaatct ctagcagtag tagttcatgt catcttatta ttcagtattt
     4441 ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa
     4501 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4561 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc
     4621 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4681 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4741 ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata
     4801 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     4861 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     4921 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg
     4981 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5041 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5101 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5161 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     5221 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5281 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     5341 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5401 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     5461 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5521 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5581 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5641 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5701 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5761 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5821 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5881 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5941 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6001 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6061 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     6121 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6181 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6241 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     6301 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6361 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6421 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6481 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6541 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6601 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6661 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6721 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6781 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6841 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6901 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6961 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     7021 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7081 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7141 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7201 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     7261 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     7321 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     7381 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     7441 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     7501 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     7561 acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca
     7621 ctaaagggaa caaaagctgg agctgcaagc ttaa
//
