LOCUS       sequence-391650-        6370 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Site directed zebrafish mutagenesis. sgRNA GAAGATCGGCCACTACATTC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6370)
  AUTHORS   Miles LB, Calcinotto V, Sonntag C, Lee C, Bryson-Richardson RJ
  TITLE     CRIMP: A CRISPR/Cas9 Insertional Mutagenesis Protocol and the CRIMP 
            Toolkit
  JOURNAL   bioRxiv 2023
REFERENCE   2  (bases 1 to 6370)
  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: "bioRxiv 
            2023"
COMMENT     Sequence Label: pSA_1_mTagBFP2-T2A-sGFP1-10_synCoTC
FEATURES             Location/Qualifiers
     source          1..6370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    446..479
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     protein_bind    533..580
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             1503..1544
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1545..1598
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(4149..4165)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4149..4165)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4162..4184)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4173..4189
                     /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(4197..4203,4204..4221,4222..4227))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4242..4263
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4380..4397)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4551..5139)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4631..4650)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5310..6101,6102..6170))
                     /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     5933..5952
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6171..6275)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6343..6361
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa
      421 tgcatctaga tgaagttcct attccgaagt tcctattctt caaatagtat aggaacttcg
      481 aagatcggcc actacattct ggcctaggag gctgctgcgg ttccagaggt gggaagttcc
      541 tattccgaag ttcctattct ctagaaagta taggaacttc aataaaagtt aatttaagcg
      601 ttcattacaa taaattcaag tgaatctgtt ttaagctagc ctttaacgtt tttaaattcc
      661 ttcaaacaaa ttggatcctc tttgcagttc ctcatttctc ggtgagctgg tgctttaatc
      721 cctttacttt ttcttcctct aggggaggag gaggatctgg aggaggagga tctggaggag
      781 gaggatctat gtccgaactt atcaaagaaa atatgcatat gaagctgtac atggagggaa
      841 ctgtggacaa ccaccacttc aagtgcacct ctgagggaga gggaaagcct tatgagggaa
      901 cccagaccat gcgcatcaag gtggtggagg gaggacctct gccttttgct tttgacatcc
      961 tggccacctc tttcctgtat ggaagcaaga ccttcatcaa ccacacccag ggaatccctg
     1021 acttcttcaa gcagtctttc cctgagggat tcacctggga aagagtgacc acctacgagg
     1081 atggaggagt gctgaccgcc acccaggaca cctctctcca ggatggatgc ctgatctaca
     1141 acgtgaagat cagaggagtg aactttacta gcaacggacc tgtgatgcaa aagaagaccc
     1201 tgggatggga ggctttcact gagaccctgt acccagctga tggaggactg gagggaagaa
     1261 atgacatggc tctgaagctg gtgggaggat ctcacctgat cgccaatgcc aagaccacct
     1321 acagaagcaa gaagcctgcc aagaacctga agatgcctgg agtgtactat gtggactaca
     1381 gactggagcg catcaaggag gccaacaatg agacctatgt ggagcagcat gaggtggctg
     1441 tggccagata ttgtgacctc cctagcaagc tgggacacaa gctgaatgga ggaggaggat
     1501 ctggaaagcc aatcccaaac cctctgctgg gactggacag caccgaggga agaggatctc
     1561 tcctgacctg tggagatgtg gaggagaacc ctggcccaat gagcaaggga gaggagctgt
     1621 ttactggagt ggtgccaatc ctggtggagc tggatggaga cgtgaatgga cacaaattct
     1681 ctgtgagagg agagggagag ggagatgcca ccattggaaa gctgaccctg aagttcattt
     1741 gtaccaccgg aaagctgcct gtcccttggc caaccctggt gaccaccctg acctatggag
     1801 tgcagtgctt cagcagatac cctgaccaca tgaagagaca tgacttcttc aagtctgcca
     1861 tgcctgaggg atatgtgcag gagagaacca tctctttcaa ggatgatgga aagtacaaga
     1921 ccagagctgt ggtgaagttt gagggagaca ccctggtgaa cagaattgag ctgaaaggca
     1981 ctgacttcaa ggaggatgga aacatcctgg gacacaagct ggagtacaac ttcaactctc
     2041 acaatgtgta catcaccgct gataagcaga agaatggcat caaggccaac ttcaccgtga
     2101 gacacaatgt ggaggatgga tctgtgcagc tggctgatca ctatcagcag aacaccccaa
     2161 ttggagacgg acctgtgctg ctgcctgata atcattacct gagcacccag actgtgctga
     2221 gcaaggaccc aaatgagaag taaactagta ttaagtctca gccaccgtta actgaacatg
     2281 tcaaaacctg tggagactgt tgagatttga tgttctgaaa agataaagcc tataaataaa
     2341 acaatactca aatacaaaag gtatcttttt taaaaagtta gtttactgct tccttaggct
     2401 ctttctaaat cccacccacc tgaaggacat caattatgga tttatccttc atgccttgta
     2461 taaatacaga tgtgcatatg aatttttatg ggttttttaa aaatgtggtt cattctatat
     2521 acataacaat tactttcaac atacataagc ctacctgtat ttgtgcatta tattacatgg
     2581 tacagataaa tagtcatgtg ccccatgaac attttggtca aagatagatt acagatacaa
     2641 tggtggtccc ataagattat gatgccatat ttttactgta cctcttcttg tgcctagata
     2701 cacaaatact gacttgtgtt ataattgcct gcagtattca ataatatggt gtatagattt
     2761 gtagcctacg agctataacc tatactatat agccaaggag tgcaggaggg tatgccatct
     2821 agatttgtgt gagtacactg atgtttgaac aataaaatca cctaatgata ggtttctcaa
     2881 accatatccc agtccttaag caacacatga ctaatggatt caaccatttc ccctgttcgt
     2941 tcagattgct ttttcttttt gcccctacaa ttttgttgat aatactgttt tttggggctt
     3001 ttgtttttgt tttttttttt ttttttgaga caagagtctc actctgtcgt caggctggag
     3061 tgcaatggcg ctatctcagc tcactgcaac ctctgcctcc caggttcaag cgattctcct
     3121 gcctcagcct cctgagtagc tgagattaca ggcatgcgcc accgcgcccg gctaattttg
     3181 tatttttagt agagacgcag tttcaccatg ttggtcaggc tggtcttgaa cacccgacct
     3241 caggtgatcc acccgcctca gcttcccaaa gtgctgggat taaaggcgtg agccaccaca
     3301 cctggccaat ttactgtttt ataccatgct tttttgtgtg tattctaggt tccctaaagc
     3361 aaacatgcat tttaaatttt gatagaacca gatgacatac aaaagtgcct gttttctctg
     3421 cattattaaa actgaaggta atcagtcttt tatttctatg actccaactg gcaaaaaata
     3481 tttgccattt taactttttt tttttgagac ggagtcaaaa ggtgccagtt ttctctgcat
     3541 tattaaaact gaaggtaatc agtcttttat ttctatgact ccaactggca aaagatattt
     3601 gccacgttaa cttttttttt tttttttttg agacggaggc tcacactgtc acccaggctg
     3661 aagtgcagtg gcatgatctc agctcacttg caacctccgc ctcccaggtt caagtgattc
     3721 tcctgcctca gcctcccaag gagttgggat tacaggcgca tacccggctt attttttgta
     3781 tttttagtag aaacgggatt tcaccgtgtt agccaggatg gtctcaatct ccagacttcg
     3841 tgatcgccca cctcggcctc ccaaagtgct gggattacag gcgtgagcca ccgcgcccgg
     3901 ccctcagtag gcgattttta tatgtggtat aaagtagaat tcttacagat ggatacccgt
     3961 ttatgccagt gccatgactg aaaccctgct tgtccaactt gaaatatctt caatgcccat
     4021 ctattcttcc ttgatcttgt tttttcctgt gataatacaa tagtttttac cagttaatca
     4081 gttaagatga ggcttaccaa atatatggga gtactactga acacgatatc aagcttggcg
     4141 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4201 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4261 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4321 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4381 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4441 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4501 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4561 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4621 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4681 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4741 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4801 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4861 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4921 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4981 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5041 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5101 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5161 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5221 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5281 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5341 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5401 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5461 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5521 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5581 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5641 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5701 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5761 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5821 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5881 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5941 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6001 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6061 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6121 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6181 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6241 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6301 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     6361 ccctttcgtc
//
