LOCUS       sequence-478949-        9723 bp    DNA     circular SYN 17-JUL-2025
DEFINITION  Expression of array of 48 crRNAs targeting EFNA1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9723)
  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 9723)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 17, 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-48xEFNA1 array
FEATURES             Location/Qualifiers
     source          1..9723
                     /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          2986..4002
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3926..3948
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4010..4029
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     misc_feature    6113..6701
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6166..6186)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6584..6595)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6776..7009
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(7128..7146)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7214..7318
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7319..7387,7388..8179)
                     /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(7537..7556)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8350..8938
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8839..8858
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9092..9109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9349..9370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9385..9390,9391..9408,9409..9415)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9423..9439
                     /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     9428..9450
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9447..9463
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9447..9463
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9513..9532
                     /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 gggggggcgc gcgccaggcg gggggggggg gggggggggg gggggggggg
     2761 gggggggggg gggggggggg ggcgccccag gggggggggg gggggcggag agttcttttt
     2821 tttggggggg ggggcggggg gggggccttc aaaaagaaaa ggcggggcgg ggcgaggcgg
     2881 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
     2941 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
     3001 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     3061 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     3121 cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc
     3181 cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg
     3241 gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg
     3301 gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg
     3361 tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga
     3421 gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt
     3481 gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc
     3541 gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc
     3601 ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg
     3661 gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg
     3721 tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc
     3781 ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg
     3841 ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc
     3901 cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctagc
     3961 gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg
     4021 gttattgtgc tgtctcatca ttttggcaaa gggcggccgc atcttgtgga aaggacgaaa
     4081 caccgtaatt tctactaagt gtagatcccg gacccactga gtagattaat ttctactaag
     4141 tgtagatcct ttacaacaga agacatttaa tttctactaa gtgtagataa aggagcaact
     4201 ggtagaacta atttctacta agtgtagata agggagtgta atgcttcatt aatttctact
     4261 aagtgtagat cccttctcag agcaccccaa taatttctac taagtgtaga tgagatggca
     4321 gagagaaatg ttaatttcta ctaagtgtag atcataaccc ccctgatact tataatttct
     4381 actaagtgta gatgtgatac caagctgccc acttaatttc tactaagtgt agattcttaa
     4441 acccatcctt acactaattt ctactaagtg tagatccaag gcccctgtgt tcatgtaatt
     4501 tctactaagt gtagatgcgc tatgagcaca aagatataat ttctactaag tgtagatgtg
     4561 ctgtccccag tattctctaa tttctactaa gtgtagatcc cctactggga ccccacccta
     4621 atttctacta agtgtagata actcccaata tgtactgatt aatttctact aagtgtagat
     4681 gttcccccat ggtctgttct taatttctac taagtgtaga tgatgggctt ctacctattt
     4741 ctaatttcta ctaagtgtag atcatgacct ctcacatttc actaatttct actaagtgta
     4801 gatcatgtta taaacccttt gtataatttc tactaagtgt agatggtatc tgaccacact
     4861 gacataattt ctactaagtg tagatctccc ctcacttctc cgtcataatt tctactaagt
     4921 gtagatattc atctacatcc caatgttaat ttctactaag tgtagatgat aagtcaaaga
     4981 gcgctaataa tttctactaa gtgtagatcc attacataca tgcttgggta atttctacta
     5041 agtgtagatt ctctaaaagc cacacttcct aatttctact aagtgtagat ccatgatctc
     5101 cctctcccag taatttctac taagtgtaga tcaataaaca tagagtacca ataatttcta
     5161 ctaagtgtag ataaagcctc tctctgctcc tctaatttct actaagtgta gatagggggc
     5221 tgaatgaatt gattaatttc tactaagtgt agatacttgc cccagggccc tgcataattt
     5281 ctactaagtg tagatgagta ataggggtgc cagagtaatt tctactaagt gtagataata
     5341 cagggtgaat gcataataat ttctactaag tgtagatatt gcaatggcaa actctgataa
     5401 tttctactaa gtgtagatac ttaaaggact ggggcattta atttctacta agtgtagatg
     5461 ggaggggaag gccagagctt aatttctact aagtgtagat gtacccgaaa gatactgagt
     5521 taatttctac taagtgtaga tcctcacagc cctaaacctg ctaatttcta ctaagtgtag
     5581 atctctgagg aaattcctgc cctaatttct actaagtgta gatatgtgtg gttacagttt
     5641 gtctaatttc tactaagtgt agataaggac caacaggaga cagttaattt ctactaagtg
     5701 tagatctgga ttctgattac agaattaatt tctactaagt gtagatcgtt cccaagctcc
     5761 caggtttaat ttctactaag tgtagattgc ccatgccagg tgttgactaa tttctactaa
     5821 gtgtagattg ggtagaagag aatgataata atttctacta agtgtagatc agaaaggact
     5881 catggaggat aatttctact aagtgtagat ctcaaaagtg aagctagtat taatttctac
     5941 taagtgtaga tgccgggaca gttatcccgg ttaatttcta ctaagtgtag atgacaatgt
     6001 gataaacatg actaatttct actaagtgta gatccagatg gggagcaaga gtctaatttc
     6061 tactaagtgt agatagaaca gttatgtcca gcagcgagaa tcgatagatc ctaatcaacc
     6121 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     6181 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     6241 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     6301 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     6361 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     6421 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     6481 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     6541 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     6601 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     6661 ccctcagacg agtcggatct ccctttgggc cgcctccccg cctgagatcc tttaagacca
     6721 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     6781 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg
     6841 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     6901 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     6961 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca
     7021 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
     7081 cccgggttaa ttaaggaaag ggctagatca ttcttgaaga cgaaagggcc tcgtgatacg
     7141 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     7201 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     7261 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     7321 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     7381 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     7441 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     7501 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     7561 tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     7621 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     7681 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     7741 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     7801 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     7861 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     7921 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     7981 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     8041 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     8101 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     8161 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     8221 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     8281 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     8341 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     8401 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     8461 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     8521 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     8581 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     8641 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     8701 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     8761 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     8821 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     8881 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     8941 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     9001 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     9061 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     9121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagcaagct
     9181 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     9241 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctccccgtg
     9301 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     9361 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     9421 aattgtgagc ggataacaat ttcacacagg aaacagctat gacatgatta cgaatttcac
     9481 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     9541 ttatcatgtc tggatcaact ggataactca agctaaccaa aatcatccca aacttcccac
     9601 cccataccct attaccactg ccaattacct gtggtttcat ttactctaaa cctgtgattc
     9661 ctctgaatta ttttcatttt aaagaaattg tatttgttaa atatgtacta caaacttagt
     9721 agt
//
