LOCUS       sequence-192151-        7658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FISSEQ-hgRNA-Design2 in a lentiviral backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7658)
  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 7658)
  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-Design2
FEATURES             Location/Qualifiers
     source          1..7658
                     /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    2263..2380
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2429..2939
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2858..2876
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3029..4054
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3029..4054
                     /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(4078..4094)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4079..4095)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             4177..4410
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4482..4603
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4519..4538)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4573..4592
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4643..4778
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4798..4817)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4799..4817)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4827..4844)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4827..4843)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4836..4858)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4985..5440
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5072..5091)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5282..5303
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5466..5570
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5571..5639,5640..6431)
                     /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(5789..5808)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6602..7190
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7091..7110
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7344..7361
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7478..7499
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7514..7519,7520..7537,7538..7544)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7552..7568
                     /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     7557..7579
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7576..7592
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7576..7592
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7611..7631
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7613..7631
                     /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 acttattaca
     2161 ctgttgtctt ataccaactt tcctaataag tggcaccgag tcggtgcttt ttttcctgca
     2221 ggaattctcg acctcgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2281 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2341 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2401 cagagatcca ctttggccgc ggctcgaggg ggttggggtt gcgccttttc caaggcagcc
     2461 ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg
     2521 accctgggtc tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta
     2581 cccttgtggg ccccccggcg acgcttcctg ctccgcccct aagtcgggaa ggttccttgc
     2641 ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg tctcactagt accctcgcag
     2701 acggacagcg ccagggagca atggcagcgc gccgaccgcg atgggctgtg gccaatagcg
     2761 gctgctcagc agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt
     2821 gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc
     2881 cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat caccgacctc tctccccagg
     2941 gggatccacc ggatcaagct tgccagctgg ggcgccctct ggtaaggttg ggaagccctg
     3001 cagatctgat cccggggggc aatgagatat gaaaaagcct gaactcaccg cgacgtctgt
     3061 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     3121 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     3181 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
     3241 gctcccgatt ccggaagtgc ttgacattgg ggaatttagc gagagcctga cctattgcat
     3301 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
     3361 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
     3421 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     3481 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     3541 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     3601 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     3661 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     3721 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     3781 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
     3841 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     3901 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     3961 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     4021 acgccccagc actcgtccga gggcaaagga atagagtaga tgccgaccgg gatctatcga
     4081 taccgtcgac ctcgaggggg ggcccggtac ctttaagacc aatgacttac aaggcagctg
     4141 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
     4201 gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct
     4261 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     4321 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     4381 ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt attattcagt
     4441 atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt
     4501 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4561 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc
     4621 tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     4681 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     4741 gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc
     4801 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     4861 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     4921 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     4981 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     5041 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     5101 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     5161 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     5221 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5281 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     5341 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     5401 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     5461 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5521 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5581 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5641 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5701 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5761 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5821 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5881 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5941 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     6001 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6061 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6121 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6181 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6241 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     6301 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6361 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6421 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6481 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6541 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6601 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6661 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6721 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6781 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6841 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6901 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6961 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     7021 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7081 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7141 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7201 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     7261 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     7321 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     7381 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     7441 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     7501 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     7561 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
     7621 ctcactaaag ggaacaaaag ctggagctgc aagcttaa
//
