LOCUS       sequence-234742-        4145 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To clone sgRNA spacer sequence for microinjections. This sgRNA 
            tracrRNA contains 2x com stem loops.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4145)
  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 4145)
  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-com-Ds
FEATURES             Location/Qualifiers
     source          1..4145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    116..137
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(152..157,158..175,176..182)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    190..206
                     /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     195..217
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     214..230
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     214..230
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     249..269
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        251..269
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1581..1600)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1582..1600)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1610..1627)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1610..1626)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1619..1641)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1768..2223
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1855..1874)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2065..2086
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2249..2353
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2354..2422,2423..3214)
                     /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(2572..2591)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3385..3973
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3874..3893
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4127..4144
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
       61 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
      121 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
      181 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
      241 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctcccc gactagggat
      301 gaaaacggtc ggtaacggtc ggtaaaatac ctctaccgtt ttcattttca tatttaactt
      361 gcgggacgga aacgaaaacg ggatataccg gtaacgaaaa cgaacgggat aaatacggta
      421 atcgaaaacc gatacgatcc ggtcgggtta aagtcgaaat cggacgggaa ccggtatttt
      481 tgttcggtaa aatcacacat gaaaacatat attcaaaact taaaaacaaa tataaaaaat
      541 tgtaaacaca agtcttaatt aaacatagat aaaatccata taaatctgga gcacacatag
      601 tttaatgtag cacataagtg ataagtcttg ggctcttggc taacataaga agccatataa
      661 gtctacttct agaccatggg aattcgatat ctgcttcgcc gcaagcttgc tagcaaaaaa
      721 gcaccgactc ggtgccactt ggccctgcag gatgctcgca ggcattcagt ggccaagttg
      781 ataacggact agccttattt taacttgcta ggccctgcag gatgctcgca ggcattcagt
      841 ggcctagctc taaaacagag acggtcgaca gtctgcagtg tcgtctctcg aaccaagagc
      901 tggagggaga gctatatata ccagggactt ctgggtatgt ttttgggagg tggtgagtga
      961 ctaaaccact tattcagctc ccttagatca agtctgaccc tatatcatgg tgacataaac
     1021 ctgcaaactg ataaaacctg aaagatctca aatccagagt ttgtgtgagg gattaccgtg
     1081 gtaattttca aactgtaaag catatgcaaa attatctggt cttggcttga gtgattgggt
     1141 gtctcggtgt gatgcaggga cgttttcagt gacgtgtcct tctccctccc ccacaggcat
     1201 gcgcagaaca tttcccccct ccttgaagac cagaacaaaa gacgccgaga gcaggaaact
     1261 cgtcttactg aatgaccgag gctggagaaa tacgtaaact cgagtcgagg gatctgttgc
     1321 agtcatcccg aattagaaaa tacggtaacg aaacgggatc atcccgatta aaaacgggat
     1381 cccggtgaaa cggtcgggaa actagctcta ccgtttccgt ttccgtttac cgttttgtat
     1441 atcccgtttc cgttccgttt tcgtttttta cctcgggttc gaaatcgatc gggataaaac
     1501 taacaaaatc ggttatacga taacggtcgg tacgggattt tcccatccta ctttcatccc
     1561 tgcgtcgggt acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     1621 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     1681 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     1741 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     1801 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1861 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1921 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1981 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     2041 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     2101 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     2161 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     2221 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2281 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2341 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     2401 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     2461 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     2521 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     2581 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     2641 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     2701 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     2761 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     2821 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     2881 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     2941 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     3001 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3061 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     3121 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3181 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     3241 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3301 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3361 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3421 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3481 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     3541 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3601 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3661 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3721 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3781 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3841 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3901 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3961 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4021 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4081 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4141 cgagg
//
