LOCUS       sequence-234784-        4095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To clone sgRNA spacer sequence for microinjections in zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4095)
  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 4095)
  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:sgRNA-Ds
FEATURES             Location/Qualifiers
     source          1..4095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    92..113
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(128..133,134..151,152..158)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    166..182
                     /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     171..193
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     190..206
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     190..206
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     225..245
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        227..245
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1507..1526)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1508..1526)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1536..1553)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1536..1552)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1545..1567)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1694..2149
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1781..1800)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1991..2012
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2175..2279
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2280..2348,2349..3140)
                     /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(2498..2517)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3311..3899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3800..3819
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4053..4070
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
       61 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
      121 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
      181 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
      241 aaagggaaca aaagctggag ctccccgact agggatgaaa acggtcggta acggtcggta
      301 aaatacctct accgttttca ttttcatatt taacttgcgg gacggaaacg aaaacgggat
      361 ataccggtaa cgaaaacgaa cgggataaat acggtaatcg aaaaccgata cgatccggtc
      421 gggttaaagt cgaaatcgga cgggaaccgg tatttttgtt cggtaaaatc acacatgaaa
      481 acatatattc aaaacttaaa aacaaatata aaaaattgta aacacaagtc ttaattaaac
      541 atagataaaa tccatataaa tctggagcac acatagttta atgtagcaca taagtgataa
      601 gtcttgggct cttggctaac ataagaagcc atataagtct acttctagac catgggaatt
      661 cgatatctgc ttcgccgcaa gcttgctagc aaaaaagcac cgactcggtg ccactttttc
      721 aagttgataa cggactagcc ttatttaaac ttgctatgct gtttccagca tagctcttaa
      781 acagagacgg tcgacagtct gcagtgtcgt ctctcgaacc aagagctgga gggagagcta
      841 tatataccag ggacttctgg gtatgttttt gggaggtggt gagtgactaa accacttatt
      901 cagctccctt agatcaagtc tgaccctata tcatggtgac ataaacctgc aaactgataa
      961 aacctgaaag atctcaaatc cagagtttgt gtgagggatt accgtggtaa ttttcaaact
     1021 gtaaagcata tgcaaaatta tctggtcttg gcttgagtga ttgggtgtct cggtgtgatg
     1081 cagggacgtt ttcagtgacg tgtccttctc cctcccccac aggcatgcgc agaacatttc
     1141 ccccctcctt gaagaccaga acaaaagacg ccgagagcag gaaactcgtc ttactgaatg
     1201 accgaggctg gagaaatacg taaactcgag tcgagggatc tgttgcagtc atcccgaatt
     1261 agaaaatacg gtaacgaaac gggatcatcc cgattaaaaa cgggatcccg gtgaaacggt
     1321 cgggaaacta gctctaccgt ttccgtttcc gtttaccgtt ttgtatatcc cgtttccgtt
     1381 ccgttttcgt tttttacctc gggttcgaaa tcgatcggga taaaactaac aaaatcggtt
     1441 atacgataac ggtcggtacg ggattttccc atcctacttt catccctgcg tcgggtaccc
     1501 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     1561 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     1621 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     1681 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1741 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1801 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1861 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1921 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1981 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2041 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2101 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     2161 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2221 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     2281 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     2341 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     2401 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     2461 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     2521 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     2581 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     2641 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     2701 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2761 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2821 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2881 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2941 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     3001 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3061 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3121 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     3181 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3241 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3301 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3361 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3421 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     3481 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3541 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3601 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3661 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3721 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3781 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3841 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3901 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3961 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4021 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4081 agcgcccaat acgca
//
