LOCUS       sequence-310823-        8268 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  making transgenics expressing a UAS driven YFP and NTR 2.0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8268)
  AUTHORS   Sharrock AV, Mulligan TS, Hall KR, Williams EM, White DT, Zhang L, 
            Emmerich K, Matthews F, Nimmagadda S, Washington S, Le KD, Meir-Levi
            D, Cox OL, Saxena MT, Calof AL, Lopez-Burks ME, Lander AD, Ding D, 
            Ji H, Ackerley DF, Mumm JS
  TITLE     NTR 2.0: a rationally engineered prodrug-converting enzyme with 
            substantially enhanced efficacy for targeted cell ablation.
  JOURNAL   Nat Methods. 2022 Feb;19(2):205-215. doi: 
            10.1038/s41592-021-01364-4. Epub 2022 Feb 7.
  PUBMED    35132245
REFERENCE   2  (bases 1 to 8268)
  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: 
            "10.1038/s41592-021-01364-4"; journalName: "Nat Methods"; date: 
            "2022-02"; volume: "19"; issue: "2"; pages: "205-215"
COMMENT     Sequence Label: 5xUAS:GAP-tagYFP-P2A-NfsB_Vv F70A/F108Y,he:tag...
FEATURES             Location/Qualifiers
     source          1..8268
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..26
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     18..35
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     19..35
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     42..61
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        42..60
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(73..84,85..272)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     protein_bind    complement(350..383)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             695..1411
                     /codon_start=1
                     /product="monomeric yellow fluorescent protein"
                     /label=TagYFP
                     /note="mammalian codon optimized"
                     /translation="MVSKGEELFAGIVPVLIELDGDVHGHKFSVRGEGEGDADYGKLEI
                     KFICTTGKLPVPWPTLVTTLTYGVQCFARYPKHMKMNDFFKSAMPEGYIQERTILFQDD
                     GKYKTRGEVKFEGDTLVNRIELKGKDFKEDGNILGHKLEYSFNSHNVYITPDKANNGLE
                     VNFKTRHNIEGGGVQLADHYQTNVPLGDGPVLIPINHYLSYQTDISKDRNEARDHMVLL
                     ESVSACSHTHGMDELYR"
     CDS             1433..1489
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     intron          2175..2747
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     polyA_signal    2824..2945
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2835..2854)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2889..2908
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    complement(3274..3307)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      3787..3796
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4875..4892)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4881..5106
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    join(5226..5388,5389..5400)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     promoter        complement(5451..5469)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5452..5469)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5487..5503)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5487..5503)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5500..5522)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5511..5527
                     /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(5535..5541,5542..5559,5560..5565))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5580..5601
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5718..5735)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5889..6477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5969..5988)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6648..7439,7440..7508))
                     /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     7271..7290
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7509..7613)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7691..8146)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7828..7849)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8040..8059
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactatagg
       61 gcgaattggg cccagaggtg taaagtactt gagtaatttt acttgattac tgtacttaag
      121 tattattttt ggggattttt actttacttg agtacaatta aaaatcaata cttttacttt
      181 tacttaatta cattttttta gaaaaaaaag tactttttac tccttacaat tttatttaca
      241 gtcaaaaagt acttattttt tggagatcac ttcattctat tttcccttgc tattaccaaa
      301 ccaattgaat tgcgctgatg cccagtttaa tttagggtaa tggccggcca taacttcgta
      361 tagcatacat tatacgaagt tatccggagt actgtcctcc gacactagag gtcggagtac
      421 tgtcctccga cgcaaggcgg agtactgtcc tccgacgcaa ggtcggagta ctgtcctccg
      481 acactagagg tcggagtact gtcctccgcc atacgtagca gggtagactg tagagggtat
      541 ataatggaac ccatcgcgtc tcagcctcac tttgagctcc tccacacgat ttcaccaggc
      601 cgccaccatg ctgtgttgta tcagaagaac aaagcctgtg gagaagaatg aggaggctga
      661 ccaggaactg cagcctgcag gaagcacagc tacaatggtt agcaaaggcg aggagctgtt
      721 cgccggcatc gtgcccgtgc tgatcgagct ggacggcgac gtgcacggcc acaagttcag
      781 cgtgcgcggc gagggcgagg gcgacgccga ctacggcaag ctggagatca agttcatctg
      841 caccaccggc aagctgcccg tgccctggcc caccctggtg accaccctca cctacggcgt
      901 acagtgcttc gcccgctacc ccaagcacat gaagatgaac gacttcttca agagcgccat
      961 gcccgagggc tacatccagg agcgcaccat cctcttccaa gacgacggca agtacaagac
     1021 ccgcggcgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcaa
     1081 ggacttcaag gaggacggca acatcctggg ccacaagctg gagtacagct tcaacagcca
     1141 caacgtctac atcacccccg acaaggccaa caacggcctg gaggtgaact tcaagacccg
     1201 ccacaacatc gagggcggcg gcgtgcagct ggccgaccac taccagacca acgtgcccct
     1261 gggcgacggc cccgtgctga tccccatcaa ccactacctg agctaccaga ccgacatcag
     1321 caaggaccgc aacgaggccc gcgaccacat ggtgctcctg gagtccgtca gcgcctgcag
     1381 ccacacccac ggcatggacg agctgtaccg ctacttgtac aagggaagcg gagctactaa
     1441 cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cccgggccta tgactattgt
     1501 tcaagctgcc caatcccgct actccaccaa agcctttgat gcttcgcgca aattgcctga
     1561 agagaaagtc gcggcagtga aagagttaat ccgcatgagt gcgtccagtg tcaactcgca
     1621 accttggcat tttattgtcg cgagcagtga agagggaaaa gcgcgcatcg ccaaagcaac
     1681 acaaggtggt tttgctgcta atgagcgcaa gattttggat gcttctcacg ttgtggtgtt
     1741 ctgcgccaaa acggcgattg atgaagcgta cctactcgac cttttggaaa gcgaagacaa
     1801 agatggccgc tacgccgatg tcgaagcaaa aaatggcatg cacgctggtc gttcattttt
     1861 cgtcaacatg caccgctttg acttgaaaga cgcgcatcac tggatggaaa agcaagttta
     1921 cctcaatgtg gggacgctgc tattgggtgc ttctgcgatg gagatcgacg cggtgccaat
     1981 tgaaggcttc gatgccaagg tgcttgatga ggagtttggt ctgcgtgaga agggctttac
     2041 cagcgtggtg attgtgccgc tgggttacca tagcgaagac gattttaatg ctaagctgcc
     2101 aaaatcacgt tggtcagcag agactgtttt taccgaaatc taagtcgagc acgaccgatc
     2161 ctgagaactt cagggtgagt ttggggaccc ttgattgttc tttctttttc gctattgtaa
     2221 aattcatgtt atatggaggg ggcaaagttt tcagggtgtt gtttagaatg ggaagatgtc
     2281 ccttgtatca ccatggaccc tcatgataat tttgtttctt tcactttcta ctctgttgac
     2341 aaccattgtc tcctcttatt ttcttttcat tttctgtaac tttttcgtta aactttagct
     2401 tgcatttgta acgaattttt aaattcactt ttgtttattt gtcagattgt aagtactttc
     2461 tctaatcact tttttttcaa ggcaatcagg gtatattata ttgtacttca gcacagtttt
     2521 agagaacaat tgttataatt aaatgataag gtagaatatt tctgcatata aattctggct
     2581 ggcgtggaaa tattcttatt ggtagaaaca actacaccct ggtcatcatc ctgcctttct
     2641 ctttatggtt acaatgatat acactgtttg agatgaggat aaaatactct gagtccaaac
     2701 cgggcccctc tgctaaccat gttcatgcct tcttctcttt cctacagctc ctgggcaacg
     2761 tgctggttgt tgtgctgtct catcattttg gcaaagaatt aattcctcga cgtccagaca
     2821 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     2881 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     2941 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     3001 ttttttgtcg cgaatctaga ttaagattga atcctgttgc cggtcttgcg atgattatca
     3061 tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat
     3121 ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa
     3181 acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag
     3241 atcgggaatt gatcccccct cgacagcaag cttataactt cgtatagcat acattatacg
     3301 aagttattca accactccag gcatagctag agccactgat gcatctatag agctgaagta
     3361 aagatagggt tgatgcaaat cgatttagaa ttttcaatgt agaaaaaaca aaataaaaag
     3421 tctattcact tgatggatag attagtcaaa caaacataga gtaggcactt cccttgactc
     3481 tgtaaacttt tggatttaac agattgtgca ctccttttag ttaaacacat tagtccctga
     3541 aaactaagac cctaaccata atcccattgt agacccacct actcgggtga atataaagct
     3601 cgcacagatc atctcacaga catcagacct tggcaaagga gcatcctgaa catctctaca
     3661 cactgaggca accctagggc cgccaccatg ctgtgttgta tcagaagaac aaagcctgtg
     3721 gagaagaatg aggaggctga ccaggaactg cagagcacgg taccgcggtc cctgtatcca
     3781 cctgtcgcca ccatggtgag caagggagag gagctgttca caggagtggt gcctatcctg
     3841 gtggagctgg atggagatgt gaatggacac aagttctctg tgagaggaga gggagaggga
     3901 gatgctacaa atggaaagct gacactgaag ttcatctgca caacaggaaa gctgcctgtg
     3961 ccttggccta cactggtgac aacactgagc catggagtgc agtgttttgc tagataccct
     4021 gaccatatga agcagcatga cttcttcaag tctgctatgc ctgagggata tgtgcaggag
     4081 agaacaatct tcttcaagga tgatggaaca tacaagacaa gagctgaggt gaagtttgag
     4141 ggagacacac tggtgaacag aattgaactg aagggagtgg acttcaagga ggatggaaac
     4201 atcctgggac acaagctgga gtacaacttc aacagccaca acatctacat tatggctgtg
     4261 aagcagaaga atggaatcaa ggtgaacttc aagatcagac acaatgtgga ggatggatct
     4321 gtgcagctgg ctgaccacta ccagcagaac acacctattg gagatggacc tgtgctgctg
     4381 cctgacagcc actacctgag cacacagtct gtgctgagca aggaccctaa tgagaagaga
     4441 gaccatatgg tgctgctgga gttcagaaca gctgctggaa tcacactggg aatggacgag
     4501 ctgtacaagt aaagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg
     4561 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     4621 caattgttgt tgttatcgaa ttctgcagga attgggtggc atccctgtga cccctcccca
     4681 gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa
     4741 ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg
     4801 gtggtatgga gcaaggggca agttgggaag acaacctgta gggcacgagg gggggcccga
     4861 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     4921 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     4981 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     5041 ggacagcaag ggggaggatt gggaagacaa tagcaaggca tgctggggat gcggtgggct
     5101 ctatggtagg gataacaggg taatataaaa taaaataaaa taaaataaaa taaaataaaa
     5161 taaaataaaa taaaataaag cgcgcactag tgatatcaag cttaaacaag aatctctagt
     5221 tttctttctt gcttttactt ttacttcctt aatactcaag tacaatttta atggagtact
     5281 tttttacttt tactcaagta agattctagc cagatacttt tacttttaat tgagtaaaat
     5341 tttccctaag tacttgtact ttcacttgag taaaattttt gagtactttt tacacctctg
     5401 ctcgaccata tgggagagct cccaacgcgt tggatgcata gcttgagtat tctatagtgt
     5461 cacctaaata gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5521 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5581 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5641 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5701 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5761 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5821 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5881 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5941 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     6001 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6061 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     6121 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6181 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6241 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6301 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     6361 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6421 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6481 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6541 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6601 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6661 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6721 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6781 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6841 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6901 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6961 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     7021 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     7081 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     7141 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7201 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7261 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7321 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7381 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7441 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7501 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7561 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7621 ccccgaaaag tgccacctga tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     7681 ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     7741 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     7801 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     7861 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     7921 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     7981 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     8041 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     8101 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat tcgccattca
     8161 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     8221 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggt
//
