LOCUS       sequence-234963-        4083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construct with scrambled-3 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:scrambled3-sgRNA-Ds
FEATURES             Location/Qualifiers
     source          1..4083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     52..74
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     66..83
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     67..83
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     93..112
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        93..111
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1362..1382)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1362..1380)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1401..1417)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1401..1417)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1414..1436)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1425..1441
                     /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(1449..1455,1456..1473,1474..1479))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1494..1515
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1632..1649)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1803..2391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1883..1902)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2562..3353,3354..3422))
                     /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     3185..3204
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3423..3527)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3553..4008)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3690..3711)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3902..3921
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
       61 gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg
      121 gtacccgacg cagggatgaa agtaggatgg gaaaatcccg taccgaccgt tatcgtataa
      181 ccgattttgt tagttttatc ccgatcgatt tcgaacccga ggtaaaaaac gaaaacggaa
      241 cggaaacggg atatacaaaa cggtaaacgg aaacggaaac ggtagagcta gtttcccgac
      301 cgtttcaccg ggatcccgtt tttaatcggg atgatcccgt ttcgttaccg tattttctaa
      361 ttcgggatga ctgcaacaga tccctcgact cgagtttacg tatttctcca gcctcggtca
      421 ttcagtaaga cgagtttcct gctctcggcg tcttttgttc tggtcttcaa ggagggggga
      481 aatgttctgc gcatgcctgt gggggaggga gaaggacacg tcactgaaaa cgtccctgca
      541 tcacaccgag acacccaatc actcaagcca agaccagata attttgcata tgctttacag
      601 tttgaaaatt accacggtaa tccctcacac aaactctgga tttgagatct ttcaggtttt
      661 atcagtttgc aggtttatgt caccatgata tagggtcaga cttgatctaa gggagctgaa
      721 taagtggttt agtcactcac cacctcccaa aaacataccc agaagtccct ggtatatata
      781 gctctccctc cagctcttgg ttcgactcga cgacgcacgt gtgcgtttaa gagctatgct
      841 ggaaacagca tagcaagttt aaataaggct agtccgttat caacttgaaa aagtggcacc
      901 gagtcggtgc ttttttgcta gcaagcttgc ggcgaagcag atatcgaatt cccatggtct
      961 agaagtagac ttatatggct tcttatgtta gccaagagcc caagacttat cacttatgtg
     1021 ctacattaaa ctatgtgtgc tccagattta tatggatttt atctatgttt aattaagact
     1081 tgtgtttaca attttttata tttgttttta agttttgaat atatgttttc atgtgtgatt
     1141 ttaccgaaca aaaataccgg ttcccgtccg atttcgactt taacccgacc ggatcgtatc
     1201 ggttttcgat taccgtattt atcccgttcg ttttcgttac cggtatatcc cgttttcgtt
     1261 tccgtcccgc aagttaaata tgaaaatgaa aacggtagag gtattttacc gaccgttacc
     1321 gaccgttttc atccctagtc ggggagctcc agcttttgtt ccctttagtg agggttaatt
     1381 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     1441 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     1501 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1561 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1621 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1681 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1741 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1801 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1861 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1921 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1981 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2041 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2101 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2161 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2221 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2281 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2341 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2401 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2461 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2521 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2581 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2641 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2701 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2761 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2821 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2881 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2941 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3001 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3061 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3121 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3181 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3241 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3301 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3361 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3421 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3481 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3541 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     3601 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     3661 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     3721 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     3781 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     3841 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     3901 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     3961 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat
     4021 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     4081 tgg
//
