LOCUS       sequence-478699-        9610 bp    DNA     circular SYN 15-JUL-2025
DEFINITION  Expression of array of 48 crRNAs targeting HSPA1A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9610)
  AUTHORS   Yang LZ, Min YH, Liu YX, Gao BQ, Liu XQ, Huang Y, Wang H, Yang L, 
            Liu ZJ, Chen LL
  TITLE     CRISPR-array-mediated imaging of non-repetitive and multiplex 
            genomic loci in living cells.
  JOURNAL   Nat Methods. 2024 Sep;21(9):1646-1657. doi: 
            10.1038/s41592-024-02333-3. Epub 2024 Jul 4.
  PUBMED    38965442
REFERENCE   2  (bases 1 to 9610)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-024-02333-3"; journalName: "Nat Methods"; date: 
            "2024-09"; volume: "21"; issue: "9"; pages: "1646-1657"
COMMENT     Sequence Label: pCAG-48xHSPA1A array
FEATURES             Location/Qualifiers
     source          1..9610
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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"
     misc_feature    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2216..2595
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          2873..3889
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3813..3835
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3897..3916
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     misc_feature    6000..6588
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6053..6073)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6471..6482)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6663..6896
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(7015..7033)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7101..7205
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7206..7274,7275..8066)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7424..7443)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8237..8825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8726..8745
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8979..8996
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9236..9257
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9272..9277,9278..9295,9296..9302)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9310..9326
                     /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     9315..9337
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9334..9350
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9334..9350
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9400..9419
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactaggaca
     2221 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     2281 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     2341 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     2401 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     2461 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     2521 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     2581 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
     2641 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
     2701 gcgggggggg ggggggggcg cgccrggggg gggggggggg gggggggggg gggggggggg
     2761 grggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
     2821 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
     2881 tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct
     2941 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
     3001 taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg aaagccttga
     3061 ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt gcgtgtgtgt
     3121 gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc gctgcgggcg
     3181 cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg ggggcggtgc
     3241 cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg tgtgcgtggg
     3301 ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc acccccctcc
     3361 ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc gtggcgcggg
     3421 gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc
     3481 tcgggccggg gggggctcgg gggaggggcg cggcggcccc cggagcgccg gcggctgtcg
     3541 aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg cgcagggact
     3601 tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca ccccctctag
     3661 cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg agggccttcg
     3721 tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc cgcgggggga
     3781 cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg
     3841 ctctagcgcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc tcctgggcaa
     3901 cgtgctggtt attgtgctgt ctcatcattt tggcaaaggg cggccgcatc ttgtggaaag
     3961 gacgaaacac cgtaatttct actaagtgta gatcctacat gatcaaagag aattaatttc
     4021 tactaagtgt agatatatgc acacctgcac acactaattt ctactaagtg tagatcctgc
     4081 ataccctagc acagataatt tctactaagt gtagatgcct gagggtacag aaaaagtaat
     4141 ttctactaag tgtagatact accataagag ataaatgtaa tttctactaa gtgtagatgg
     4201 gtcccaacac cacatctata atttctacta agtgtagata gtgaagcctc tgtggactat
     4261 aatttctact aagtgtagat gtaggtcctt ccccatctcc taatttctac taagtgtaga
     4321 tggagaatct ccagccagac ataatttcta ctaagtgtag atgttgtatg aaagttacaa
     4381 aataatttct actaagtgta gatgagtcac tacagggtct gcataatttc tactaagtgt
     4441 agatggacac tccttccatt ccattaattt ctactaagtg tagatacaag gaaagcagtt
     4501 gtgtataatt tctactaagt gtagattgct atccagagag cacccataat ttctactaag
     4561 tgtagatgag atgaacacgt cgaaacttaa tttctactaa gtgtagatgc tcgatgcctc
     4621 tctcatgata atttctacta agtgtagatc aggccagtct acagaggaat aatttctact
     4681 aagtgtagat ccatagcgtc taaaatggaa taatttctac taagtgtaga tgtcttgtaa
     4741 atcacctagt ataatttcta ctaagtgtag attgagggag cgatccttac gttaatttct
     4801 actaagtgta gatagaccaa tcttacacaa acataatttc tactaagtgt agattgtctg
     4861 tccctgaaac cccttaattt ctactaagtg tagatctgtt gcactttcct ccctctaatt
     4921 tctactaagt gtagatctga caaggagagg gtcacctaat ttctactaag tgtagatagg
     4981 ctaccctccc aggtgtttaa tttctactaa gtgtagataa agacagcacc aaggatcata
     5041 atttctacta agtgtagata ttgctgtcta aggtgaatat aatttctact aagtgtagat
     5101 acctaaaaac ggtaacagcc taatttctac taagtgtaga tgaaccagag ttgacggttg
     5161 ctaatttcta ctaagtgtag atcccactcc atgccacagt agtaatttct actaagtgta
     5221 gatcaaggag gagggaatta ctttaatttc tactaagtgt agattagttc ctcaaacagt
     5281 agtttaattt ctactaagtg tagatcatta agctgaaagc agctgtaatt tctactaagt
     5341 gtagatccta cagccagctc agcgcctaat ttctactaag tgtagatcat tgtggccaga
     5401 gtccacataa tttctactaa gtgtagatga aggatcagag ctcagagtta atttctacta
     5461 agtgtagata catcacatac tacagctgtt aatttctact aagtgtagat ttggggagtg
     5521 tccctaagaa taatttctac taagtgtaga ttgggtggca ctgcattctc ttaatttcta
     5581 ctaagtgtag atcttagtat gtaggccgtc cttaatttct actaagtgta gatctttcag
     5641 acgcctaaca aagtaatttc tactaagtgt agattcagca gaatcagaga agtataattt
     5701 ctactaagtg tagatagttt atccaaatga acttataatt tctactaagt gtagatcctc
     5761 cccattccta gcttggtaat ttctactaag tgtagatgca ggtggccctc tttaagctaa
     5821 tttctactaa gtgtagatac ctacaaggat agaacaacta atttctacta agtgtagatc
     5881 tataggacct cctctcccct aatttctact aagtgtagat aggatgacaa gaactcttga
     5941 taatttctac taagtgtaga tactccgtgt ctggaatgct ccgagaatcg atagatccta
     6001 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     6061 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     6121 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     6181 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     6241 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     6301 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     6361 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     6421 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     6481 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     6541 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct gagatccttt
     6601 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg
     6661 actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt gtactgggtc
     6721 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     6781 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     6841 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtag
     6901 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag
     6961 tgagaggccc gggttaatta aggaaagggc tagatcattc ttgaagacga aagggcctcg
     7021 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     7081 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     7141 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     7201 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     7261 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     7321 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     7381 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     7441 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     7501 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     7561 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     7621 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     7681 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     7741 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     7801 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     7861 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     7921 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     7981 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     8041 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     8101 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     8161 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     8221 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     8281 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     8341 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     8401 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     8461 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     8521 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     8581 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     8641 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     8701 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     8761 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     8821 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     8881 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     8941 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     9001 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     9061 gcaagctcat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     9121 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     9181 ccccgtggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     9241 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     9301 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac atgattacga
     9361 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     9421 atgtatctta tcatgtctgg atcaactgga taactcaagc taaccaaaat catcccaaac
     9481 ttcccacccc ataccctatt accactgcca attacctgtg gtttcattta ctctaaacct
     9541 gtgattcctc tgaattattt tcattttaaa gaaattgtat ttgttaaata tgtactacaa
     9601 acttagtagt
//
