LOCUS       sequence-234961-        4083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construct with scrambled-4 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:scrambled4-sgRNA-Ds
FEATURES             Location/Qualifiers
     source          1..4083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    87..108
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(123..128,129..146,147..153)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    161..177
                     /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     166..188
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     185..201
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     185..201
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     220..240
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        222..240
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1490..1509)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1491..1509)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1519..1536)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1519..1535)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1528..1550)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1677..2132
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1764..1783)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1974..1995
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2158..2262
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2263..2331,2332..3123)
                     /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(2481..2500)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3294..3882
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3783..3802
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4036..4053
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
       61 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
      121 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
      181 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
      241 gaacaaaagc tggagctccc cgactaggga tgaaaacggt cggtaacggt cggtaaaata
      301 cctctaccgt tttcattttc atatttaact tgcgggacgg aaacgaaaac gggatatacc
      361 ggtaacgaaa acgaacggga taaatacggt aatcgaaaac cgatacgatc cggtcgggtt
      421 aaagtcgaaa tcggacggga accggtattt ttgttcggta aaatcacaca tgaaaacata
      481 tattcaaaac ttaaaaacaa atataaaaaa ttgtaaacac aagtcttaat taaacataga
      541 taaaatccat ataaatctgg agcacacata gtttaatgta gcacataagt gataagtctt
      601 gggctcttgg ctaacataag aagccatata agtctacttc tagaccatgg gaattcgata
      661 tctgcttcgc cgcaagcttg ctagcaaaaa agcaccgact cggtgccact ttttcaagtt
      721 gataacggac tagccttatt taaacttgct atgctgtttc cagcatagct cttaaactat
      781 tcgtatagag tatagatcga accaagagct ggagggagag ctatatatac cagggacttc
      841 tgggtatgtt tttgggaggt ggtgagtgac taaaccactt attcagctcc cttagatcaa
      901 gtctgaccct atatcatggt gacataaacc tgcaaactga taaaacctga aagatctcaa
      961 atccagagtt tgtgtgaggg attaccgtgg taattttcaa actgtaaagc atatgcaaaa
     1021 ttatctggtc ttggcttgag tgattgggtg tctcggtgtg atgcagggac gttttcagtg
     1081 acgtgtcctt ctccctcccc cacaggcatg cgcagaacat ttcccccctc cttgaagacc
     1141 agaacaaaag acgccgagag caggaaactc gtcttactga atgaccgagg ctggagaaat
     1201 acgtaaactc gagtcgaggg atctgttgca gtcatcccga attagaaaat acggtaacga
     1261 aacgggatca tcccgattaa aaacgggatc ccggtgaaac ggtcgggaaa ctagctctac
     1321 cgtttccgtt tccgtttacc gttttgtata tcccgtttcc gttccgtttt cgttttttac
     1381 ctcgggttcg aaatcgatcg ggataaaact aacaaaatcg gttatacgat aacggtcggt
     1441 acgggatttt cccatcctac tttcatccct gcgtcgggta cccaattcgc cctatagtga
     1501 gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     1561 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     1621 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc
     1681 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1741 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1801 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1861 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1921 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1981 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2041 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2101 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     2161 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2221 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     2281 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     2341 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     2401 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     2461 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     2521 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     2581 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     2641 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     2701 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2761 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     2821 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     2881 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2941 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     3001 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     3061 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     3121 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     3181 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3241 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3301 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3361 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3421 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     3481 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3541 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3601 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3661 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3721 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3781 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3841 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3901 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3961 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4021 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     4081 ccg
//
