LOCUS       sequence-463679-        7689 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of cre inducible and her4.3 (Notch) dependent Cas9-GFP 
            and U6-driven 4 sgRNAs for SABER-seq in zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7689)
  AUTHORS   Siniscalco AM, Perera RP, Greenslade JE, Veeravenkatasubramanian H, 
            Masters A, Doll HM, Raj B
  TITLE     Barcoding Notch signaling in the developing brain.
  JOURNAL   Development. 2024 Nov 22:dev.203102. doi: 10.1242/dev.203102.
  PUBMED    39575683
REFERENCE   2  (bases 1 to 7689)
  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: 
            "Development. 2024 Nov 22:dev.203102. doi: 10.1242/dev.203102."
COMMENT     Sequence Label: p160Tol2-her4.3:switchCas9
FEATURES             Location/Qualifiers
     source          1..7689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    159..293
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(183..202)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     237..256
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(732..919,920..931)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(965..981)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(965..981)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1018..1038)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1018..1036)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1057..1073)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1070..1092)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1081..1097
                     /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(1105..1111,1112..1129,1130..1135))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1150..1171
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1288..1305)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1459..2047)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1539..1558)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2218..3009,3010..3078))
                     /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     2841..2860
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3079..3183)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3346..3367)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(3716..3727,3728..3890)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     4263..4279
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4299..4319
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    6716..6740
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     6741..6758
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6742..6758
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6768..6787
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6768..6786
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(6825..6858)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      6865..6874
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6871..7548
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(7051..7071)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     7463..7486
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
ORIGIN
        1 tccttctccc tcccccacag gcatgcgcag aacatttccc ccctccttga agaccagaac
       61 aaaagacgcc gagagcagga aactcgtctt actgaatgac cgaggctgga gaaacgctga
      121 gagacttact gatgcatttc atttaatgta tcgatgatga tccagacatg ataagataca
      181 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
      241 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
      301 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
      361 agtaaaacct ctacaaatgt ggtatggctg attatgatcc tctagatcag atctgcgaag
      421 atacggccac gggtgctctt gatcctgtgg ctgattttgg actgtgctgc tcgcagctgc
      481 tgatgaatca catacttcct ccattttctt ccactgattg actgttataa tttccctaat
      541 ttccaggtca aggtgctgtg cattgtggta atagatgtga catgacgtca cttccaaagg
      601 accaatgaac atgtctgacc aatttcatat aatgtgaaaa cgattttcat aggcagaata
      661 aataacattt aaattaaact gggcatcagc gcaattcaat tggtttggta atagcaaggg
      721 aaaatagaat gaagtgatct ccaaaaaata agtacttttt gactgtaaat aaaattgtaa
      781 ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat
      841 ttttaattgt actcaagtaa agtaaaaatc cccaaaaata atacttaagt acagtaatca
      901 agtaaaatta ctcaagtact ttacacctct ggttcttgac cccctacctt cagcaagccc
      961 agcagatcca ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct
     1021 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     1081 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     1141 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     1201 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     1261 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     1321 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     1381 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     1441 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     1501 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     1561 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     1621 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     1681 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     1741 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     1801 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     1861 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     1921 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     1981 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     2041 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     2101 acgttaaggc attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     2161 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     2221 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     2281 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     2341 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     2401 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     2461 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     2521 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     2581 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     2641 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     2701 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     2761 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     2821 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     2881 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     2941 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     3001 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     3061 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     3121 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     3181 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     3241 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     3301 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     3361 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     3421 gatggcccac gctggctaag aactcatcag cctccccggt ccatctaccc acgtaccaat
     3481 gcaccaattg gccacaatga cggctactac atggtgccat tccttcctct ttataggaat
     3541 ggagactacc tcctgtccaa caatgctctt ggatacgagt acgcctacct gttggaccca
     3601 ggtcattgca caacaccaga aatgccctct gatctgcaaa agacgtgaat atctgttcag
     3661 acacccatat ccactctgtt ccacacaggt cagaggtttg tccaggagtt cttgacagag
     3721 gtgtaaaaag tactcaaaaa ttttactcaa gtgaaagtac aagtacttag ggaaaatttt
     3781 actcaattaa aagtaaaagt atctggctag aatcttactt gagtaaaagt aaaaaagtac
     3841 tccattaaaa ttgtacttga gtattaagga agtaaaagta aaagcaagaa agaaaactag
     3901 agattcttgt ttaagctttt aatctcaaaa aacattaaat gaaatgcata caaggtttta
     3961 tcctgcttta gaactgtttg tatttaatta tcaaactata agacagacaa tctaatgcca
     4021 gtacacgcta ctcaaagttg taaaacctca gatttaactt cagtagaagc tgattctcaa
     4081 aattgttagt gtcaagccta gctcttttgg ggctgaaaag caatcctgca gtgctgaaaa
     4141 gcctctcaca ggcagccgat gcgggaagag gtgtattagt cttgatagag aggctgcaaa
     4201 tagcaggaaa cgtgagcaga gactccctgg tgtctgaaac acaggccaga tgggccctcg
     4261 agcaggaaac agctatgacc atgattacgc caagctatca actttgtata gaaaagttgt
     4321 tttgcattat ttccctaatt ttaaatgtct gtgacattgt ttaatatttc ccagttcaat
     4381 ggaactgcag atgtaatgag gacttcttcc tgattctgaa atatgtatgt acattttgtt
     4441 actgatgtta ctgctttgct atgttaaagc acccatcctt catagttgag gcaaatgtga
     4501 attgttagat ttgctcacat gaagaaaagc aatcatattg catatggcaa ataaactgta
     4561 caatcaactt tcttgttttg ggttttcttt aactctcaac tgtttcaaca aaaactcaca
     4621 tttttaaaga atatgtcatt ttcaaaaaag tttttaaagt caaacaaatg tttgaccgtc
     4681 ataagaaaat cagcttttgg acatctaaaa agaggtgtat aatgttagac acgtgtatgt
     4741 tttaacagta tatttataga ataatggcct ctcaagaaat tagttcatta ttaagaaatt
     4801 actttttaaa aaactttagt ccttcatatg ttgaaattca cattcaaata tttatatttc
     4861 atactgaatt taatgcagat aaatccaaga ctacgaggga taattttatt gcccttatga
     4921 catgttgttt tcagaaatca taactttcaa aacattcaaa aatttcttcc ctactgattt
     4981 ttaagaagtt ctgttacctg aaaaatatat tttgttaagc aacagtaaat ccattgaatt
     5041 cagcgtacac ccttgagtct tttaaaaatc taaatatgac actttcctga ctatagtcta
     5101 cacaataaga tagatatgaa tgcaaaactt gactagattt tagtttttac tctcacaaaa
     5161 aatgcaaagc agtacatttt tgacctaaaa aaattttatt taaataaaat cctctatttc
     5221 tttcccacac caataaagca catgtataag atgatgaata tgcatgctaa aacagagacc
     5281 tcaatagagc actatatcca cattaaacaa gaccttgttt ttctggaatt aatagctcaa
     5341 acccatgatg aggcgacttt ctaaacgaag cagtgactga tggttaaagc tactgagagc
     5401 tcattccctg gagttttccc acactctgcc cattcaaaca ctcaacaaaa gtgtttcatt
     5461 agaaacaatt caacacatgg ccataaggac ttcaacagag accccctcgt gagagactct
     5521 actgaatctg gactatttgt cctaattaag atgtatttag agcatttggt gattgcactt
     5581 ctaaagttta ttgttcaata aaaacagaaa taagatcaaa ctagaccgtg tttcatgtta
     5641 cactcaaact gatgcttcaa attgtgctta ggctatgtaa aattcagact catggcttat
     5701 gagatatact ttctgttatt tgactcaaaa gcaaaacact gtgcaagatg ttttattgta
     5761 atgctgcacc gttttaaagt ttgagggctc actttaggtt tatatttttc ttgggatggc
     5821 agagcttaat ttgtagcgtc catagtgttc agtgtcacgt ggtcctttag aaattatttt
     5881 attgtgctga tttgctgctc caaaaatatt tctcattatt acctgtgtgg aaaacagtag
     5941 tgcaacagat tcatttagta attaactttt tgtaaataaa aaagtttgaa aaggacacca
     6001 tttatttgaa atataaatta tctagaacgg tataaatgca tttgctgtca cttttgtaca
     6061 agcatcctta ataaataaaa gttttaatta tttcaaaaat gcccataaaa aaacttaaac
     6121 tgtttccaag tgccctcagg aacaagctaa aataccccag gggctctaca cgtgcctgct
     6181 ttatcatctg aataacttgc ttacatttag ttttttttac aaaaaatatt acaacaattt
     6241 aacagaacag ctaccataaa ctgctcacat attcaccaga tatgaaacct catgtttgtt
     6301 ttaaaataaa aaaaatcaaa ctaagtcttg atgtgtttta ctgatgggaa ggttcctgta
     6361 ttaatggcac gtgctttgtc aataaatgac cccagtatca gccaacaaac aaagctttaa
     6421 tcgaggggtc tccactttct cacacactct ccatcctcaa cagccaaccc tccacctgct
     6481 ccccaaagac cagcccattg tcccctgagc aataagcacg tcatttagca tgtggcggcc
     6541 ctctgctcct tctgtcttta gagctctccc attggctgca gactgtgaga acctccagca
     6601 aactccagac tcacacattg atatggagat ttgtgagtat aaatagagga gagaggagtg
     6661 agagtttagc acagctcaga tctgatcctg aaggagaact gaacacaaga cacacacaag
     6721 tttgtacaaa aaagcaggct tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac
     6781 tatagggcga attgggtacc gggccccccc tcgagggccg gcctataact tcgtatagca
     6841 tacattatac gaagttatcc ggtcgccacc atggcctcct ccgagaacgt catcaccgag
     6901 ttcatgcgct tcaaggtgcg catggagggc accgtgaacg gccacgagtt cgagatcgag
     6961 ggcgagggcg agggccgccc ctacgagggc cacaacaccg tgaagctgaa ggtgaccaag
     7021 ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc agttccagta cggctccaag
     7081 gtgtacgtga agcaccccgc cgacatcccc gactacaaga agctgtcctt ccccgagggc
     7141 ttcaagtggg agcgcgtgat gaactttgag gacggcggcg tggcgaccgt gacccaggac
     7201 tcctccctgc aggacggctg cttcatctac aaggtgaagt tcatcggcgt gaacttcccc
     7261 tccgacggcc ccgtgatgca gaagaagacc atgggctggg aggcctccac cgagcgcctg
     7321 tacccccgcg acggcgtgct gaagggcgag acccacaagg ccctgaagct gaaggacggc
     7381 ggccactacc tggtggagtt caagtccatc tacatggcca agaagcccgt gcagctgccc
     7441 ggctactact acgtggacgc caagctggac atcacctccc acaacgagga ctacaccatc
     7501 gtggagcagt acgagcgcac cgagggccgc caccacctgt tcctgtagcg gccgcgactc
     7561 tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     7621 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     7681 gcagcttat
//
