LOCUS       sequence-234737-        4145 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To clone sgRNA spacer sequence for microinjections. This sgRNA 
            tracrRNA contains 2x MS2 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-MS2-Ds
FEATURES             Location/Qualifiers
     source          1..4145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(466..484)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        complement(536..554)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     primer_bind     complement(1296..1315)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1297..1315)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1325..1342)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1325..1341)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1334..1356)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1483..1938
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1570..1589)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1780..1801
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1964..2068
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2069..2137,2138..2929)
                     /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(2287..2306)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3100..3688
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3589..3608
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3842..3859
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3976..3997
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4012..4017,4018..4035,4036..4042)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4050..4066
                     /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     4055..4077
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4074..4090
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4074..4090
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4109..4129
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4111..4129
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tccccgacta gggatgaaaa cggtcggtaa cggtcggtaa aatacctcta ccgttttcat
       61 tttcatattt aacttgcggg acggaaacga aaacgggata taccggtaac gaaaacgaac
      121 gggataaata cggtaatcga aaaccgatac gatccggtcg ggttaaagtc gaaatcggac
      181 gggaaccggt atttttgttc ggtaaaatca cacatgaaaa catatattca aaacttaaaa
      241 acaaatataa aaaattgtaa acacaagtct taattaaaca tagataaaat ccatataaat
      301 ctggagcaca catagtttaa tgtagcacat aagtgataag tcttgggctc ttggctaaca
      361 taagaagcca tataagtcta cttctagacc atgggaattc gatatctgct tcgccgcaag
      421 cttgctagca aaaaagcacc gactcggtgc cacttggccc tgcagacatg ggtgatcctc
      481 atgttggcca agttgataac ggactagcct tattttaact tgctaggccc tgcagacatg
      541 ggtgatcctc atgttggcct agctctaaaa cagagacggt cgacagtctg cagtgtcgtc
      601 tctcgaacca agagctggag ggagagctat atataccagg gacttctggg tatgtttttg
      661 ggaggtggtg agtgactaaa ccacttattc agctccctta gatcaagtct gaccctatat
      721 catggtgaca taaacctgca aactgataaa acctgaaaga tctcaaatcc agagtttgtg
      781 tgagggatta ccgtggtaat tttcaaactg taaagcatat gcaaaattat ctggtcttgg
      841 cttgagtgat tgggtgtctc ggtgtgatgc agggacgttt tcagtgacgt gtccttctcc
      901 ctcccccaca ggcatgcgca gaacatttcc cccctccttg aagaccagaa caaaagacgc
      961 cgagagcagg aaactcgtct tactgaatga ccgaggctgg agaaatacgt aaactcgagt
     1021 cgagggatct gttgcagtca tcccgaatta gaaaatacgg taacgaaacg ggatcatccc
     1081 gattaaaaac gggatcccgg tgaaacggtc gggaaactag ctctaccgtt tccgtttccg
     1141 tttaccgttt tgtatatccc gtttccgttc cgttttcgtt ttttacctcg ggttcgaaat
     1201 cgatcgggat aaaactaaca aaatcggtta tacgataacg gtcggtacgg gattttccca
     1261 tcctactttc atccctgcgt cgggtaccca attcgcccta tagtgagtcg tattacgcgc
     1321 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     1381 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     1441 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     1501 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     1561 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     1621 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     1681 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     1741 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1801 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1861 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1921 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1981 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     2041 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     2101 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     2161 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     2221 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     2281 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     2341 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     2401 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     2461 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     2521 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     2581 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     2641 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     2701 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     2761 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     2821 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     2881 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     2941 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     3001 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     3061 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3121 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3181 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3241 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3301 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3361 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3421 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3481 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3541 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3601 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3661 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3721 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3781 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3841 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     3901 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     3961 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     4021 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     4081 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
     4141 ggagc
//
