LOCUS       sequence-99003-e        5759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for CRISPR/Cas9 mediated insertion of E2A-KalTA4; to be used in 
            combination with a eGFP specific sgRNA (e.g. Plasmid 61051).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5759)
  AUTHORS   Auer TO, Duroure K, De Cian A, Concordet JP, Del Bene F
  TITLE     Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by 
            homology-independent DNA repair.
  JOURNAL   Genome Res. 2014 Jan;24(1):142-53. doi: 10.1101/gr.161638.113. Epub 
            2013 Oct 31.
  PUBMED    24179142
REFERENCE   2  (bases 1 to 5759)
  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"; doi: 
            "10.1101/gr.161638.113"; journalName: "Genome Res"; date: "2014-01";
            volume: "24"; issue: "1"; pages: "142-53"
COMMENT     Sequence Label: eGFPbait-E2A-KalTA4-pA donor vector
FEATURES             Location/Qualifiers
     source          1..5759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(393..414)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             606..665
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     CDS             675..1112
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="KLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKR
                     SPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKD
                     AVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     intron          1293..1865
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     polyA_signal    1938..2072
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1962..1981)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2016..2035
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2192..2211)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2193..2211)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2218..2235)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2218..2234)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2227..2249)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2375..2803
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2462..2481)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2672..2693
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             3147..3941
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3201..3220)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3811..3830
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(3959..4027,4028..4819)
                     /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(4177..4196)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4990..5578
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5479..5498
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5732..5749
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gcccttatag tggtaccatg gtgagcaagg
      361 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
      421 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
      481 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
      541 tgacctacgg cgtgcagtgc ttcagccgct acaagggcga attctgcaga tatccaggag
      601 gaggacagtg tactaattat gctctcttga aattggctgg agatgttgag agcaacccag
      661 gtcccggagg aggaaaactg ctctcatcca tcgagcaagc ctgcgacatt tgtcggctta
      721 agaagctgaa atgctccaag gaaaagccga aatgtgccaa atgcctgaag aacaattggg
      781 aatgtcgtta ctctcccaaa accaagcgaa gtccactcac aagggctcat ctgaccgaag
      841 tggagagcag gctagagaga ctggaacaac tctttttgct catcttccct agagaggacc
      901 ttgacatgat cctcaagatg gattctctcc aggatattaa agcccttttg actggcttat
      961 tcgtccagga caatgtgaac aaagacgctg tgacagaccg attggcaagt gtcgagaccg
     1021 atatgcctct gacactgaga cagcacagaa tcagcgctac ttcctcaagc gaagagtctt
     1081 ctaacaaggg acagagacag ctgactgttt cgagcaggtc gaccccgtcc ccggccgacg
     1141 ccctggacga cggcgacctg gacatgctgc ctgctgatgc tctcgatgat ttcgatctgg
     1201 atatgctccc ggccgacgcc ctggacgact acgacctgga catcctcccg ggtaactaag
     1261 taaggatctc gaccgatcct gagaacttca gggtgagttt ggggaccctt gattgttctt
     1321 tctttttcgc tattgtaaaa ttcatgttat atggaggggg caaagttttc agggtgttgt
     1381 ttagaatggg aagatgtccc ttgtatcacc atggaccctc atgataattt tgtttctttc
     1441 actttctact ctgttgacaa ccattgtctc ctcttatttt cttttcattt tctgtaactt
     1501 tttcgttaaa ctttagcttg catttgtaac gaatttttaa attcactttt gtttatttgt
     1561 cagattgtaa gtactttctc taatcacttt tttttcaagg caatcagggt atattatatt
     1621 gtacttcagc acagttttag agaacaattg ttataattaa atgataaggt agaatatttc
     1681 tgcatataaa ttctggctgg cgtggaaata ttcttattgg tagaaacaac tacaccctgg
     1741 tcatcatcct gcctttctct ttatggttac aatgatatac actgtttgag atgaggataa
     1801 aatactctga gtccaaaccg ggcccctctg ctaaccatgt tcatgccttc ttctctttcc
     1861 tacagctcct gggcaacgtg ctggttgttg tgctgtctca tcattttggc aaagaattaa
     1921 ttcctcgacg gatcgtagat ccagacatga taagatacat tgatgagttt ggacaaacca
     1981 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     2041 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     2101 ttcaggttca gggggaggtg tgggaggttt tttaattcgc ggccgctcta gaactagtgg
     2161 actcgagcat gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact
     2221 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     2281 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     2341 ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc
     2401 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2461 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2521 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2581 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2641 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2701 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2761 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aattcagggc
     2821 gcaagggctg ctaaaggaag cggaacacgt agaaagccag tccgcagaaa cggtgctgac
     2881 cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc gcaaagagaa
     2941 agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt ttatggacag
     3001 caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag
     3061 taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca agatctgatc
     3121 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3181 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3241 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3301 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     3361 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3421 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc ccaccttgct cctgccgaga
     3481 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3541 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     3601 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     3661 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     3721 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     3781 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     3841 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     3901 agcgcatcgc cttctatcgc cttcttgacg agttcttctg aattgaaaaa ggaagagtat
     3961 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     4021 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4081 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4141 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     4201 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     4261 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     4321 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4381 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4441 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4501 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4561 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     4621 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     4681 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     4741 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     4801 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4861 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4921 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4981 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5041 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5101 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5161 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5221 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5281 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5341 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5401 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5461 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5521 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5581 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     5641 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     5701 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaag
//
