LOCUS       sequence-366106-        7253 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to generate the Tg(QUAS:NLS-GFP; he1.1:CFP)c682 transgenic 
            line.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7253)
  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 7253)
  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-GFP-he1.1:CFP
FEATURES             Location/Qualifiers
     source          1..7253
                     /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             join(1410..1412,1413..1415,1416..2129)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1410..1412,1413..1415,1416..2126)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     protein_bind    complement(2131..2155)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    2155..2289
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     regulatory      2830..2839
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2836..2838,2839..2841,2842..3555)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3918..3935)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3924..4149
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(4150..4170)
                     /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(4178..4194)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    4210..4344
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(4783..4970,4971..4982)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5016..5032)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5016..5032)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5069..5089)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5069..5087)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5108..5124)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5121..5143)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5132..5148
                     /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(5156..5162,5163..5180,5181..5186))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5201..5222
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5339..5356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5510..6098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5590..5609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6269..7060,7061..7129))
                     /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     6892..6911
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7130..7234)
                     /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 ggtaaatcga tcgaattcaa
     1381 ggcctctcga gcctctagaa ggtggcggaa tggtgagcaa gggcgaggag ctgttcaccg
     1441 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1501 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1561 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     1621 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1681 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1741 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1801 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     1861 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     1921 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1981 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     2041 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2101 ctctcggcat ggacgagctg tacaagtaac acccagcttt cttgtacaaa gtgggatcca
     2161 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     2221 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     2281 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
     2341 gaggtttttt tcaaccactc caggcatagc tagagccact gatgcatcta tagagctgaa
     2401 gtaaagatag ggttgatgca aatcgattta gaattttcaa tgtagaaaaa acaaaataaa
     2461 aagtctattc acttgatgga tagattagtc aaacaaacat agagtaggca cttcccttga
     2521 ctctgtaaac ttttggattt aacagattgt gcactccttt tagttaaaca cattagtccc
     2581 tgaaaactaa gaccctaacc ataatcccat tgtagaccca cctactcggg tgaatataaa
     2641 gctcgcacag atcatctcac agacatcaga ccttggcaaa ggagcatcct gaacatctct
     2701 acacactgag gcaaccctag ggccgccacc atgctgtgtt gtatcagaag aacaaagcct
     2761 gtggagaaga atgaggaggc tgaccaggaa ctgcagagca cggtaccgcg ggcccgggat
     2821 ccaccggccg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     2881 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     2941 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     3001 gtgccctggc ccaccctcgt gaccaccctg acctggggcg tgcagtgctt cagccgctac
     3061 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     3121 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     3181 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     3241 aacatcctgg ggcacaagct ggagtacaac tacatcagcc acaacgtcta tatcaccgcc
     3301 gacaagcaga agaacggcat caaggccaac ttcaagatcc gccacaacat cgaggacggc
     3361 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     3421 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     3481 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     3541 gagctgtaca agtaaagcgg ccgcgactct agatcataat cagccatacc acatttgtag
     3601 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     3661 atgcaattgt tgttgttatc gaattctgca ggaattgggt ggcatccctg tgacccctcc
     3721 ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata
     3781 aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg
     3841 ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcacg agggggggcc
     3901 cgaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     3961 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4021 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4081 gcaggacagc aagggggagg attgggaaga caatagcaag gcatgctggg gatgcggtgg
     4141 gctctatggc aactttatta tacatagttg ataattcact ggccgtcgtt ttacggtacc
     4201 atcgatgatg atccagacat gataagatac attgatgagt ttggacaaac cacaactaga
     4261 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     4321 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     4381 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct
     4441 gattatgatc ctctagatca gatctgcgaa gatacggcca cgggtgctct tgatcctgtg
     4501 gctgattttg gactgtgctg ctcgcagctg ctgatgaatc acatacttcc tccattttct
     4561 tccactgatt gactgttata atttccctaa tttccaggtc aaggtgctgt gcattgtggt
     4621 aatagatgtg acatgacgtc acttccaaag gaccaatgaa catgtctgac caatttcata
     4681 taatgtgaaa acgattttca taggcagaat aaataacatt taaattaaac tgggcatcag
     4741 cgcaattcaa ttggtttggt aatagcaagg gaaaatagaa tgaagtgatc tccaaaaaat
     4801 aagtactttt tgactgtaaa taaaattgta aggagtaaaa agtacttttt tttctaaaaa
     4861 aatgtaatta agtaaaagta aaagtattga tttttaattg tactcaagta aagtaaaaat
     4921 ccccaaaaat aatacttaag tacagtaatc aagtaaaatt actcaagtac tttacacctc
     4981 tggttcttga ccccctacct tcagcaagcc cagcagatcc actagttcta gagcggccgc
     5041 caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     5101 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5161 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5221 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5281 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5341 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5401 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5461 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5521 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5581 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5641 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5701 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5761 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5821 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5881 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5941 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6001 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6061 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6121 cggggtctga cgctcagtgg aacgaaaact cacgttaagg cattttggtc atgagattat
     6181 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6241 gtatatatga gtaaacttgg tctgacagtt accaatgctt aataagtgag gcacctatct
     6301 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6361 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6421 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6481 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6541 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6601 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6661 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6721 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6781 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6841 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6901 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6961 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7021 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7081 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7141 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7201 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtg
//
