LOCUS       sequence-439413-        7919 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPRi machinery fish codon optimized.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7919)
  TITLE     Ori lab plasmids
REFERENCE   2  (bases 1 to 7919)
  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"
COMMENT     Sequence Label: pME-dCas9-BFP-KRAB
FEATURES             Location/Qualifiers
     source          1..7919
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(2475..2495)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      2504..2513
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2549..2567)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2577..2593)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2595..2694)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2710..2726)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2719..2741)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     terminator      2790..2876
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2968..2995
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3154..3171)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3325..3913)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3405..3424)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4060..4869)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4783..4802
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4861..4880
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     4982..4998
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4982..4998
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5003..5021
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5039..5138
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     5165..5185
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5167..5185
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(5225..5410)
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             complement(6191..6211)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(6221..6241)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(6245..6271)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     RBS             6366..6374
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
ORIGIN
        1 acgtacatat ctcttccatt ctgcagatag tacagataca gcttctcatt ctgcagctgt
       61 gtgttttcca ctgggtgctc tttcaggatc tggctaccca gctccttaat gccttcctcg
      121 attcttttca tcctctctct actgttcttc tgacccttct gggttgtctg attttcgcga
      181 gccatctcaa tcacgatgtt ttcgggctta tggcccatga ctttcaccag ctcatcgacg
      241 attttcactg tctgcagaat gcctttcttg atggctgggc ttccagccag gtttgcaatc
      301 tgctcatgca gggagtggcc ctgtccagac acctgagcct tctggatgtc ctctttgaag
      361 gtcagactgt catcgtgaat cagctgcata aaattgcggt tggcgaagcc atcacttttc
      421 agaaagtcca ggattgtctt tcctgactgt ttatccctaa tgccgttgat cagcttcctg
      481 ctcagtctac cccagccagt gtagcgcctt ctcttcagct gtttcatcac cttgtcatcg
      541 aacagatgag cataggtttt cagtctttcc tcgatcattc ctctgtcctc aaacagggtc
      601 agtgtcagca caatgtcttc caggatatcc tcattttcct cgttgtccag gaagtcttta
      661 tccttaatga tcttcagcag atcgtggtaa gcgcccaggc ttgcgttaaa tctgtcttcg
      721 actccgctga tctccacgga atcgaagcat tcaattttct tgaagtagtc ctccttcagc
      781 tgcttgacag tcactttcct gttggtttta aacagcagat ccacgattgc tttcttctgc
      841 tctcctgaca ggaaggctgg ctttctcatg ccctcggtga cgtatttcac ctttgtcagt
      901 tcgttataga cagtgaaata ctcgtacagc agggagtgct taggcagcac tttctcattg
      961 ggcaggtttt tgtcaaaatt agtcattctc tcgatgaagg actgtgcaga ggcgccctta
     1021 tcgaccactt cctcgaagtt ccaaggagta atggtttcct cggatttgcg agtcatccag
     1081 gcaaatctgc tgtttccgcg agccagtgga cccacatagt aagggattct gaaggtcaga
     1141 attttttcga tcttctcgcg gttatctttc agaaaggggt agaagtcttc ctggcgcctc
     1201 aggatagcgt gcagctctcc cagatgaatc tggtgtggga tggaaccgtt atcaaatgtg
     1261 cgctgcttcc tcagcaggtc ctctctattc agtttcacca gcagttcctc agttccgtcc
     1321 atcttctcca gaataggttt gataaacttg tagaattcct cctgtgatgc gcctccgtcg
     1381 atataaccgg cgtagccgtt cttggactga tcaaagaaaa tttccttgta cttctctggc
     1441 agctgctgcc tgaccagagc cttcagcagg gtcagatcct gatggtgctc gtcatatctc
     1501 ttgatcatgc tggcactcag aggtgctttt gtaatctcgc tgttcactct cagaatatct
     1561 gacagcagga ttgcgtcgga cagatttttg gcagccagga acaggtcagc gtactgatct
     1621 ccgatctgtg ccagcaggtt atccaggtca tcgtcatatg tgtcctttga cagctgcagt
     1681 ttggcgtcct cagccagatc aaaattgctt ttgaagttag gagtcaggcc cagactcaga
     1741 gcgatcagat ttccaaacag accgttcctc ttttctccgg gcagctgtgc aatcaggttc
     1801 tccagtctgc gtgacttgga cagcctggct gacaggatag cttttgcgtc cactctggaa
     1861 gcattaatag ggttttcctc aaacagctga ttgtaaatct ggaccagctg gatgaacagc
     1921 ttgtccacat cggaattgtc ggggttcaga tcgccctcga tcagaaaatg acccctgaac
     1981 ttaatcatgt gggccagagc cagatagatc agtctcaggt cggctttatc agtagagtca
     2041 gccagtttct tgcgcagatg atagatggtt gggtattttt cgtgataggc gacctcgtcc
     2101 acaatgtttc caaagatagg atggcgctcg tgtttcttat cttcctcgac caggaaactt
     2161 tcctccagcc tatgaaagaa tgaatcgtcc accttagcca tttcgttaga gaaaatctcc
     2221 tgcaggtaac agattctatt cttccttctg gtatagcgcc ttcttgctgt tcttttcagg
     2281 cgtgttgctt cggcagtctc tccagaaccg aacagcaggg ctccaatcag atttttcttg
     2341 atgctgtgcc tgtcggtgtt acccagcacc ttaaatttct tgctagggac cttgtagtcg
     2401 tctgtaatca ctgcccaacc cacggagtta gttccaatgg ccaggccgat gctatacttc
     2461 ttatccatgc ttcccacctt tctcttcttc ttaggtggag aagccatggt ggcgtcgacc
     2521 tcgagggggg gcccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     2581 gccgtcgttt tacaagcctg cttttttgta caaagttggc attataaaaa agcattgctc
     2641 atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttggggccc
     2701 gagcttaaga ctggccgtcg ttttacaacg tcgtgactgg gaaaacatcc atgctagcgt
     2761 taacgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcggaagact
     2821 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
     2881 cgggagcgga tttgaacgtt gtgaagcaac ggcccggagg gtggcgggca ggacgcccgc
     2941 cataaactgc caggcatcaa actaagcaga aggccatcct gacggatggc ctttttgcgt
     3001 ttctacaaac tcttcctggc tagcggtacg cgtattaatt gcgttgcgct cactgcccgc
     3061 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3121 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3181 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3241 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3301 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3361 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3421 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3481 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3541 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3601 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3661 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3721 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3781 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3841 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3901 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3961 cgcgtaactc acgttaaggg attttggtca tgagcttgcg ccgtcccgtc aagtcagcgt
     4021 aatgctctgc cagtgttaca accaattaac caattctgat tagaaaaact catcgagcat
     4081 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     4141 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     4201 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     4261 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     4321 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     4381 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     4441 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     4501 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     4561 tgtttttccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     4621 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     4681 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     4741 cccatacaag cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     4801 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctcgacgttt cccgttgaat
     4861 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     4921 tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacgg gccagagctg
     4981 ccaggaaaca gctatgacca tgtaatacga ctcactatag gggatatcag ctggatggca
     5041 aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg ataagcaatg
     5101 ctttcttata atgccaactt tgtacaagaa agctgggtgc tatgaccatg attacgccaa
     5161 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctccaccgcg gtggcggccg
     5221 ctcaaggttc ctcgcctttc tcgagtctca ggatcacgtc aggcttggtc agctggtatc
     5281 ccagagacac gaggttcttg tagttctcga gcatcacgtt tctgtacacg atctgctgag
     5341 cggtgtccag cagcttccat tcctctcttg tgaagtccac gaacacgtcc ttgaaggtga
     5401 ccagggttct gctccaagct gtaaggctct tagcgtccat tccgcctccg ccaccattca
     5461 gcttgtgtcc cagcttgcta ggcaggtcac agtatctagc cacggccacc tcgtgctgct
     5521 cgacgtatgt ctcgttgttg gcctctttga ttctttccag tctgtagtcc acgtagtaca
     5581 cgccaggcat cttcaggttc ttggcgggct tcttgcttct gtaggtggtc ttgatgttgg
     5641 cgatcaggtg gcttcctccg acgagcttca gggccatgtc gtttctgcct tccagtccac
     5701 catcagcagg gtacagtgtc tcggtgaagg cttcccatcc gagggtcttt ttctgcatca
     5761 caggtccgtt gctggtgaag ttcacgcctc tgatcttcac gttgtagatc aggcatccgt
     5821 cctgcaggct tgtatcctgg gtagctgtca gcactccgcc gtcctcgtat gtggtcactc
     5881 tctcccaggt gaatccctca gggaagctct gcttgaagaa gtcggggatt ccctgagtgt
     5941 ggttgatgaa ggtcttgctg ccgtacagga agctggtagc caggatatcg aaggcgaaag
     6001 gcagaggtcc gccttccacc accttgattc tcatggtctg ggttccctcg taaggctttc
     6061 cctcgccttc gcttgtgcac ttgaagtggt ggttgtccac ggtgccttcc atgtacagct
     6121 tcatgtgcat gttttctttg atcagctcgc tagatccgct gcttccgttg cttccgcttc
     6181 cgattccgtc gacctttctc ttcttcttgg gatcttccat cacctttctc ttcttcttag
     6241 gagaggcata atcgggcacg tcatagggat aggatcccac cggtgagtcg ccacccagct
     6301 gagacaggtc aatccttgtt tcgtacaggc cggtaatact ctgatggatc agtgtagcgt
     6361 ccagcacctc cttagtggag gtgtacctct ttctatcgat tgtagtgtcg aagtacttga
     6421 aagctgcagg tgcgcccagg tttgtcagag tgaacagatg aatgatattt tcggcctgct
     6481 ccctgatggg cttatctctg tgtttgttgt aagcagacag gaccttatcc agatttgcgt
     6541 cggccaggat cactctttta gagaactcgc taatctgttc aatgatctcg tccagataat
     6601 gcttgtgctg ctccacaaac agctgtttct gttcgttatc ctctggagat cctttcagct
     6661 tctcgtagtg gcttgccaga tacaggaagt tcacgtactt agaaggcagg gccagttcat
     6721 tgcctttctg cagctctcca gcgcttgcca gcatgcgttt cctgccgttt tccagctcaa
     6781 acagactgta cttgggcagt ttaatgatca ggtctttctt cacttcctta taacctttgg
     6841 cctccagaaa atcgattggg ttcttctcga aagagcttct ctccataatt gtgataccca
     6901 gcagctcctt gacagatttc agtttcttgc ttttgccctt ctccacctta gcgaccacca
     6961 ggacggagta tgccacagtg ggagagtcaa agcctccata cttcttaggg tcccagtctt
     7021 tctttctagc gatcagctta tcagagttgc gttttggcag aatactttct tttgagaaac
     7081 cgcctgtctg cacctcagtt ttcttgacga tattcacttg aggcatgctc aggaccttcc
     7141 tcacggttgc aaaatctctg cctttgtccc acacgatttc gcctgtctct ccgtttgtct
     7201 caatcagggg ccttttgcgg atttctccat tggccagggt gatctctgtc ttaaagaaat
     7261 tcatgatgtt tgagtaaaag aaatacttgg cagtagcttt tccaatttcc tgctcggact
     7321 tggcgatcat ttttctgaca tcgtacacct tatagtctcc gtacacgaat tcgctctcca
     7381 gttttgggta tttcttgatc agtgcggtac cgaccacggc gttcaggtat gcgtcatggg
     7441 catggtgata attgttaatc tcgcggacct tataaaactg gaaatctttc ctaaagtcgc
     7501 tgaccagttt actcttcagg gtaatgacct tcacttctct gatcagtttg tcattctcat
     7561 cgtactttgt gttcatgcga ctatccagaa tctgtgcgac gtgtttagtg atctggcgtg
     7621 tttccaccag ctgcctcttg atgaatccag ctttgtccag ttctgacagt ccaccgcgct
     7681 ctgcttttgt caggttatca aacttcctct gagtgatcag tttggcattc agcagctgtc
     7741 tccagtagtt cttcattttc ttgaccactt cctcactagg gacattatct gattttcctc
     7801 tgtttttgtc ggacctggtc agcaccttgt tgtcaatgct gtcatctttg atgaaactct
     7861 gggggacgat ggcatccacg tcgtaatcag acagcctgtt aatatccagc tcctggtcc
//
