LOCUS       sequence-366083-        7199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to generate the Tg(QUAS:NLS-mApple; he1.1:CFP)c718 transgenic 
            line.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7199)
  AUTHORS   Spikol ED, Cheng J, Macurak M, Subedi A, Halpern ME
  TITLE     Genetically defined nucleus incertus neurons differ in connectivity 
            and function.
  JOURNAL   Elife. 2024 May 31;12:RP89516. doi: 10.7554/eLife.89516.
  PUBMED    38819436
REFERENCE   2  (bases 1 to 7199)
  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: "Elife."; 
            date: "2024-05-31"; volume: "12:RP89516. doi"; pages: " 
            10.7554/eLife.89516"
COMMENT     Sequence Label: pDestTol2-QUAS:NLS-mApple-he1.1:CFP
FEATURES             Location/Qualifiers
     source          1..7199
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(514..525,526..688)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     1061..1077
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1097..1117
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    1308..1332
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1344..1364
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1365..2075
                     /codon_start=1
                     /product="photostable monomeric derivative of DsRed (Shaner
                     et al., 2008)"
                     /label=mApple
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEA
                     FQTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNF
                     EDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALK
                     SEIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     1778..1797
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2077..2101)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    2101..2235
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      2776..2785
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2782..2784,2785..2787,2788..3501)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2827..2848)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3088..3107)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3435..3456
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3864..3881)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3870..4095
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(4096..4116)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(4124..4140)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    4156..4290
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(4729..4916,4917..4928)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4962..4978)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4962..4978)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5015..5035)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5015..5033)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5054..5070)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5067..5089)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5078..5094
                     /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(5102..5108,5109..5126,5127..5132))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5147..5168
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5285..5302)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5456..6044)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5536..5555)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6215..7006,7007..7075))
                     /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     6838..6857
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7076..7180)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacgc tggctaagaa
      241 ctcatcagcc tccccggtcc atctacccac gtaccaatgc accaattggc cacaatgacg
      301 gctactacat ggtgccattc cttcctcttt ataggaatgg agactacctc ctgtccaaca
      361 atgctcttgg atacgagtac gcctacctgt tggacccagg tcattgcaca acaccagaaa
      421 tgccctctga tctgcaaaag acgtgaatat ctgttcagac acccatatcc actctgttcc
      481 acacaggtca gaggtttgtc caggagttct tgacagaggt gtaaaaagta ctcaaaaatt
      541 ttactcaagt gaaagtacaa gtacttaggg aaaattttac tcaattaaaa gtaaaagtat
      601 ctggctagaa tcttacttga gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt
      661 attaaggaag taaaagtaaa agcaagaaag aaaactagag attcttgttt aagcttttaa
      721 tctcaaaaaa cattaaatga aatgcataca aggttttatc ctgctttaga actgtttgta
      781 tttaattatc aaactataag acagacaatc taatgccagt acacgctact caaagttgta
      841 aaacctcaga tttaacttca gtagaagctg attctcaaaa ttgttagtgt caagcctagc
      901 tcttttgggg ctgaaaagca atcctgcagt gctgaaaagc ctctcacagg cagccgatgc
      961 gggaagaggt gtattagtct tgatagagag gctgcaaata gcaggaaacg tgagcagaga
     1021 ctccctggtg tctgaaacac aggccagatg ggccctcgag caggaaacag ctatgaccat
     1081 gattacgcca agctatcaac tttgtataga aaagttgggg taatcgctta tcctcggata
     1141 aacaattatc ctcacgggta atcgcttatc cgctcgggta atcgcttatc ctcgggtaat
     1201 cgcttatcct tgcaagggtc gactctagag ggtatataat ggatcccatc gcgtctcagc
     1261 ctcactttga gctcctccac acgaattccc tcgacctcga agacgcgtca agtttgtaca
     1321 aaaaagcagg ctggaccatg gctccaaaga agaagcgtaa ggtaatggtg agcaagggcg
     1381 aggagaataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
     1441 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgaggcct
     1501 ttcagaccgc taagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     1561 tgtcccctca gttcatgtac ggctccaagg tctacattaa gcacccagcc gacatccccg
     1621 actacttcaa gctgtccttc cccgagggct tcaggtggga gcgcgtgatg aacttcgagg
     1681 acggcggcat tattcacgtt aaccaggact cctccctgca ggacggcgtg ttcatctaca
     1741 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
     1801 tgggctggga ggcctccgag gagcggatgt accccgagga cggcgccctg aagagcgaga
     1861 tcaagaagag gctgaagctg aaggacggcg gccactacgc cgccgaggtc aagaccacct
     1921 acaaggccaa gaagcccgtg cagctgcccg gcgcctacat cgtcgacatc aagttggaca
     1981 tcgtgtccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     2041 actccaccgg cggcatggac gagctgtaca agtaacaccc agctttcttg tacaaagtgg
     2101 gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     2161 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     2221 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     2281 gtgtgggagg tttttttcaa ccactccagg catagctaga gccactgatg catctataga
     2341 gctgaagtaa agatagggtt gatgcaaatc gatttagaat tttcaatgta gaaaaaacaa
     2401 aataaaaagt ctattcactt gatggataga ttagtcaaac aaacatagag taggcacttc
     2461 ccttgactct gtaaactttt ggatttaaca gattgtgcac tccttttagt taaacacatt
     2521 agtccctgaa aactaagacc ctaaccataa tcccattgta gacccaccta ctcgggtgaa
     2581 tataaagctc gcacagatca tctcacagac atcagacctt ggcaaaggag catcctgaac
     2641 atctctacac actgaggcaa ccctagggcc gccaccatgc tgtgttgtat cagaagaaca
     2701 aagcctgtgg agaagaatga ggaggctgac caggaactgc agagcacggt accgcgggcc
     2761 cgggatccac cggccgccac catggtgagc aagggcgagg agctgttcac cggggtggtg
     2821 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     2881 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     2941 ctgcccgtgc cctggcccac cctcgtgacc accctgacct ggggcgtgca gtgcttcagc
     3001 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     3061 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     3121 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     3181 gacggcaaca tcctggggca caagctggag tacaactaca tcagccacaa cgtctatatc
     3241 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag
     3301 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     3361 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     3421 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     3481 atggacgagc tgtacaagta aagcggccgc gactctagat cataatcagc cataccacat
     3541 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     3601 aaatgaatgc aattgttgtt gttatcgaat tctgcaggaa ttgggtggca tccctgtgac
     3661 ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc
     3721 ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg
     3781 tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag ggcacgaggg
     3841 ggggcccgaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3901 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3961 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4021 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaaggcat gctggggatg
     4081 cggtgggctc tatggcaact ttattataca tagttgataa ttcactggcc gtcgttttac
     4141 ggtaccatcg atgatgatcc agacatgata agatacattg atgagtttgg acaaaccaca
     4201 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     4261 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     4321 caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     4381 atggctgatt atgatcctct agatcagatc tgcgaagata cggccacggg tgctcttgat
     4441 cctgtggctg attttggact gtgctgctcg cagctgctga tgaatcacat acttcctcca
     4501 ttttcttcca ctgattgact gttataattt ccctaatttc caggtcaagg tgctgtgcat
     4561 tgtggtaata gatgtgacat gacgtcactt ccaaaggacc aatgaacatg tctgaccaat
     4621 ttcatataat gtgaaaacga ttttcatagg cagaataaat aacatttaaa ttaaactggg
     4681 catcagcgca attcaattgg tttggtaata gcaagggaaa atagaatgaa gtgatctcca
     4741 aaaaataagt actttttgac tgtaaataaa attgtaagga gtaaaaagta cttttttttc
     4801 taaaaaaatg taattaagta aaagtaaaag tattgatttt taattgtact caagtaaagt
     4861 aaaaatcccc aaaaataata cttaagtaca gtaatcaagt aaaattactc aagtacttta
     4921 cacctctggt tcttgacccc ctaccttcag caagcccagc agatccacta gttctagagc
     4981 ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct
     5041 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5101 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5161 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5221 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5281 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5341 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5401 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5461 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5521 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5581 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5641 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5701 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5761 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5821 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5881 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5941 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6001 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6061 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaaggcatt ttggtcatga
     6121 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6181 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaata agtgaggcac
     6241 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6301 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6361 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6421 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6481 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6541 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6601 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6661 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6721 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6781 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6841 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6901 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6961 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7021 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7081 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7141 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtg
//
