LOCUS       sequence-455502-        8038 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector with U6 driven 4 sgRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8038)
  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 8038)
  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: p103DestTol2pA2-4xU6sgRNA
FEATURES             Location/Qualifiers
     source          1..8038
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        19..123
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(124..192,193..984)
                     /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(342..361)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1155..1743
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1644..1663
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1897..1914
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2031..2052
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2067..2072,2073..2090,2091..2097)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2105..2121
                     /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     2110..2132
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2129..2145
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2164..2184
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2166..2184
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2221..2237
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2221..2237
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(2271..2282,2283..2470)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     polyA_signal    2909..3043
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2946..2965)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3000..3019
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5216..5232
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5240..5363
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(5388..5393,5394..5411,5412..5418)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             5472..6152
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(5538..5557)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             6472..6777
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     6656..6675
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(6818..6941)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6961..6977)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    join(7350..7512,7513..7524)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     7873..7894
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
       61 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
      121 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
      181 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
      241 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
      301 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
      361 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
      421 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
      481 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
      541 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
      601 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
      661 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
      721 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
      781 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
      841 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
      901 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
      961 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     1021 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     1081 atgaccaaaa tgccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     1141 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     1201 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     1261 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     1321 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     1381 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     1441 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     1501 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     1561 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     1621 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     1681 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     1741 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     1801 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     1861 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     1921 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     1981 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     2041 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     2101 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     2161 cgcgcaatta accctcacta aagggaacaa aagctggagc tccaccgcgg tggcggccgc
     2221 tctagaacta gtggatctgc tgggcttgct gaaggtaggg ggtcaagaac cagaggtgta
     2281 aagtacttga gtaattttac ttgattactg tacttaagta ttatttttgg ggatttttac
     2341 tttacttgag tacaattaaa aatcaatact tttactttta cttaattaca tttttttaga
     2401 aaaaaaagta ctttttactc cttacaattt tatttacagt caaaaagtac ttattttttg
     2461 gagatcactt cattctattt tcccttgcta ttaccaaacc aattgaattg cgctgatgcc
     2521 cagtttaatt taaatgttat ttattctgcc tatgaaaatc gttttcacat tatatgaaat
     2581 tggtcagaca tgttcattgg tcctttggaa gtgacgtcat gtcacatcta ttaccacaat
     2641 gcacagcacc ttgacctgga aattagggaa attataacag tcaatcagtg gaagaaaatg
     2701 gaggaagtat gtgattcatc agcagctgcg agcagcacag tccaaaatca gccacaggat
     2761 caagagcacc cgtggccgta tcttcgcaga tctgatctag aggatcataa tcagccatac
     2821 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     2881 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     2941 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     3001 tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcatcatcg atacattaaa
     3061 tgaaatgcat cagtaagtct ctcagcgttt ctccagcctc ggtcattcag taagacgagt
     3121 ttcctgctct cggcgtcttt tgttctggtc ttcaaggagg ggggaaatgt tctgcgcatg
     3181 cctgtggggg agggagaagg acacgtcact gaaaacgtcc ctgcatcaca ccgagacacc
     3241 caatcactca agccaagacc agataatttt gcatatgctt tacagtttga aaattaccac
     3301 ggtaatccct cacacaaact ctggatttga gatccttcag gttttatcag tttgcaggtt
     3361 tatgtcacca tgatataggg tcagacttga tctaagggag ctgaataagt ggtttagtca
     3421 ctcaccacct cccaaaaaca tacccagaag tccctggtat atatagctct ccctccagct
     3481 cttggttcgg actggaggac ttctgggggt ttaagagcta tgctggaaac agcatagcaa
     3541 gtttaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
     3601 agtcaagtct ctcagcgttt ctccagcctc ggtcattcag taagacgagt ttcctgctct
     3661 cggcgtcttt tgttctggtc ttcaaggagg ggggaaatgt tctgcgcatg cctgtggggg
     3721 agggagaagg acacgtcact gaaaacgtcc ctgcatcaca ccgagacacc caatcactca
     3781 agccaagacc agataatttt gcatatgctt tacagtttga aaattaccac ggtaatccct
     3841 cacacaaact ctggatttga gatccttcag gttttatcag tttgcaggtt tatgtcacca
     3901 tgatataggg tcagacttga tctaagggag ctgaataagt ggtttagtca ctcaccacct
     3961 cccaaaaaca tacccagaag tccctggtat atatagctct ccctccagct cttggttcgc
     4021 cctgaagctg aaggacgggt ttaagagcta tgctggaaac agcatagcaa gtttaaataa
     4081 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt taaactcatt
     4141 accctccacg tgtctgtctg ggttttcatg ggctctgctc tagtgagagc agcttccttt
     4201 tgtcggagtg ttttgtgctc ttatccaagg caggggggat ctgcgcatgc ctgtgggggg
     4261 aggagaagga cacgtgaaca aaagctcctc gatgtcacac aggaagttca ggaactcatc
     4321 caatcactct aaagaaacgg cctgtttcct tcgcatacgc ttacagctcc aaaactctac
     4381 ggtaaaccta cataaactgc tggttttcaa attttaaaga atttaagggt ttacaggttt
     4441 actactacac agtgatttac tgacacatgt aagtgtaaat gagttgaata agtaagtaag
     4501 ccatatacca cacatgaaac acatacccag aagtcactgg tatatatagc cgtcctccag
     4561 actcccagtt cggagttcaa gtccatctac agtttaagag ctatgctgga aacagcatag
     4621 caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     4681 ttttaaacca atccgagagt ctgtgaatgt ttcgatgggc cgtttgctgg cgtctgagca
     4741 tgttgtcctc tggtctgcgg cgggggaagg tccgcgcatg cctgtggggg aaggcgaagg
     4801 acacgaggac aaaagatcag ttacgtcaca gaagaactca tccaatcaca caagcctaaa
     4861 accagcttcc tctgcatatg ctttacagtt caaaaatact acggtaaacc tctacaaaac
     4921 tgctggtttc aaactttgaa ggttttaagc gtttgcaggt ttgccccgaa gaggtttact
     4981 gtcatgtttg aggtaaatga gttgaataag taggtttatc cacttaccac atggcagaaa
     5041 catacccaga agtcccgggt ttatatagca gttctccagg ctcctagttc gctgcccggc
     5101 tactactacg gtttaagagc tatgctggaa acagcatagc aagtttaaat aaggctagtc
     5161 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttaaacctg gtaccgtaaa
     5221 acgacggcca gtgaattatc aactatgtat aataaagttg aacgagaaac gtaaaatgat
     5281 ataaatatca atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa
     5341 acacaacata tccagtcact atggcggccg cattaggcac cccaggcttt acactttatg
     5401 cttccggctc gtataatgtg tggattttga gttaggatcc gtcgagattt tcaggagcta
     5461 aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc
     5521 atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg
     5581 ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc
     5641 cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa
     5701 tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg
     5761 agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc
     5821 tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag
     5881 ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg
     5941 atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt
     6001 atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg
     6061 atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg
     6121 cggggcgtaa tctagaggat ccggcttact aaaagccaga taacagtatg cgtatttgcg
     6181 cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag
     6241 aggtatgcta tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc
     6301 aaggcatata tgatgtcaat atctccggtt cggtaagcac aaccatgcag aatgaagccc
     6361 gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc
     6421 ggtttattga aatgaacggc tcctttgccg acgagaacag gggctggtga aatgcagttt
     6481 aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat
     6541 attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca
     6601 gataaagtcc cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg
     6661 atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc
     6721 agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg
     6781 tcaggctccc ttatacacag ccagtctgca ggtcgaccat agtgactgga tatgttgtgt
     6841 tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat
     6901 atcattttac gtttctcgtt caacttttct atacaaagtt gatagcttgg cgtaatcatg
     6961 gtcatagctg tttcctgctc gagggcccat ctggcctgtg tttcagacac cagggagtct
     7021 ctgctcacgt ttcctgctat ttgcagcctc tctatcaaga ctaatacacc tcttcccgca
     7081 tcggctgcct gtgagaggct tttcagcact gcaggattgc ttttcagccc caaaagagct
     7141 aggcttgaca ctaacaattt tgagaatcag cttctactga agttaaatct gaggttttac
     7201 aactttgagt agcgtgtact ggcattagat tgtctgtctt atagtttgat aattaaatac
     7261 aaacagttct aaagcaggat aaaaccttgt atgcatttca tttaatgttt tttgagatta
     7321 aaagcttaaa caagaatctc tagttttctt tcttgctttt acttttactt ccttaatact
     7381 caagtacaat tttaatggag tactttttta cttttactca agtaagattc tagccagata
     7441 cttttacttt taattgagta aaattttccc taagtacttg tactttcact tgagtaaaat
     7501 ttttgagtac tttttacacc tctgtcaaga actcctggac aaacctctga cctgtgtgga
     7561 acagagtgga tatgggtgtc tgaacagata ttcacgtctt ttgcagatca gagggcattt
     7621 ctggtgttgt gcaatgacct gggtccaaca ggtaggcgta ctcgtatcca agagcattgt
     7681 tggacaggag gtagtctcca ttcctataaa gaggaaggaa tggcaccatg tagtagccgt
     7741 cattgtggcc aattggtgca ttggtacgtg ggtagatgga ccggggaggc tgatgagttc
     7801 ttagccagcg tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     7861 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     7921 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     7981 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggc
//
