LOCUS       sequence-234966-        4083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construct with scrambled-2 sgRNA sequence for microinjections in 
            zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4083)
  AUTHORS   Chong-Morrison V, Mayes S, Simoes FC, Senanayake U, Carroll DS, 
            Riley PR, Wilson SW, Sauka-Spengler T
  TITLE     Ac/Ds transposition for CRISPR/dCas9-SID4x epigenome modulation in 
            zebrafish.
  JOURNAL   Biol Open. 2023 Jun 15;12(6):bio059995. doi: 10.1242/bio.059995. 
            Epub 2023 Jun 27.
  PUBMED    37367831
REFERENCE   2  (bases 1 to 4083)
  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"; journalName: "Biol 
            Open."; date: "2023-06-15"; volume: "12(6):bio059995. doi"; pages: "
            10.1242/bio.059995. Epub 2023 Jun 27"
COMMENT     Sequence Label: pVC-Ds-DrU6a:scrambled2-sgRNA-Ds
FEATURES             Location/Qualifiers
     source          1..4083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..51
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     43..60
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     44..60
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     70..89
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        70..88
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1339..1359)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1339..1357)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1378..1394)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1378..1394)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1391..1413)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1402..1418
                     /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)."
     promoter        complement(join(1426..1432,1433..1450,1451..1456))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1471..1492
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1609..1626)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1780..2368)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1860..1879)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2539..3330,3331..3399))
                     /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     3162..3181
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3400..3504)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3530..3985)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3667..3688)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3879..3898
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
       61 gagcgcgcgt aatacgactc actatagggc gaattgggta cccgacgcag ggatgaaagt
      121 aggatgggaa aatcccgtac cgaccgttat cgtataaccg attttgttag ttttatcccg
      181 atcgatttcg aacccgaggt aaaaaacgaa aacggaacgg aaacgggata tacaaaacgg
      241 taaacggaaa cggaaacggt agagctagtt tcccgaccgt ttcaccggga tcccgttttt
      301 aatcgggatg atcccgtttc gttaccgtat tttctaattc gggatgactg caacagatcc
      361 ctcgactcga gtttacgtat ttctccagcc tcggtcattc agtaagacga gtttcctgct
      421 ctcggcgtct tttgttctgg tcttcaagga ggggggaaat gttctgcgca tgcctgtggg
      481 ggagggagaa ggacacgtca ctgaaaacgt ccctgcatca caccgagaca cccaatcact
      541 caagccaaga ccagataatt ttgcatatgc tttacagttt gaaaattacc acggtaatcc
      601 ctcacacaaa ctctggattt gagatctttc aggttttatc agtttgcagg tttatgtcac
      661 catgatatag ggtcagactt gatctaaggg agctgaataa gtggtttagt cactcaccac
      721 ctcccaaaaa catacccaga agtccctggt atatatagct ctccctccag ctcttggttc
      781 gggccacagg cgattatcgc cgtttaagag ctatgctgga aacagcatag caagtttaaa
      841 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgctagca
      901 agcttgcggc gaagcagata tcgaattccc atggtctaga agtagactta tatggcttct
      961 tatgttagcc aagagcccaa gacttatcac ttatgtgcta cattaaacta tgtgtgctcc
     1021 agatttatat ggattttatc tatgtttaat taagacttgt gtttacaatt ttttatattt
     1081 gtttttaagt tttgaatata tgttttcatg tgtgatttta ccgaacaaaa ataccggttc
     1141 ccgtccgatt tcgactttaa cccgaccgga tcgtatcggt tttcgattac cgtatttatc
     1201 ccgttcgttt tcgttaccgg tatatcccgt tttcgtttcc gtcccgcaag ttaaatatga
     1261 aaatgaaaac ggtagaggta ttttaccgac cgttaccgac cgttttcatc cctagtcggg
     1321 gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc
     1381 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     1441 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     1501 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     1561 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     1621 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     1681 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     1741 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     1801 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     1861 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     1921 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     1981 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2041 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2101 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2161 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     2221 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     2281 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     2341 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     2401 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     2461 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     2521 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     2581 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     2641 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     2701 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     2761 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     2821 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     2881 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     2941 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3001 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3061 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3121 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     3181 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     3241 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     3301 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     3361 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     3421 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     3481 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     3541 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3601 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3661 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3721 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     3781 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3841 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3901 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3961 cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa
     4021 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     4081 agg
//
