LOCUS       Exported                8260 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  making transgenics expressing a UAS driven mCherry and NTR 2.0.
ACCESSION   .
VERSION     .
KEYWORDS    5xUAS:GAP-mCherry-P2A-NfsB_Vv F70A/F108Y,he:tagBFP2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8260)
  AUTHORS   Sharrock AV, Mulligan TS, Hall KR, Williams EM, White DT, Zhang L, 
            Matthews F, Nimmagadda S, Washington S, Le K , 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   bioRxiv
REFERENCE   2  (bases 1 to 8260)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8260
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    329..362
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     regulatory      681..690
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             687..1394
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(832..850)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1100..1119
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1294..1317
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1404..1460
                     /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          2146..2718
                     /label=-beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     polyA_signal    2795..2916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2806..2825)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2860..2879
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    3245..3278
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     regulatory      3758..3767
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     complement(4846..4863)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4852..5077
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(5422..5440)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5423..5440)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5458..5474)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5458..5474)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5471..5493)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5482..5498
                     /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(5506..5536)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5551..5572
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5689..5706)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5860..6448)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5940..5959)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6619..7479)
                     /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     7242..7261
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7480..7584)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7662..8117)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7799..7820)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8011..8030
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8243..5
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8257..14
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8258..14
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggc ccagaggtgt
       61 aaagtacttg agtaatttta cttgattact gtacttaagt attatttttg gggattttta
      121 ctttacttga gtacaattaa aaatcaatac ttttactttt acttaattac atttttttag
      181 aaaaaaaagt actttttact ccttacaatt ttatttacag tcaaaaagta cttatttttt
      241 ggagatcact tcattctatt ttcccttgct attaccaaac caattgaatt gcgctgatgc
      301 ccagtttaat ttagggtaat ggccggccat aacttcgtat agcatacatt atacgaagtt
      361 atccggagta ctgtcctccg acactagagg tcggagtact gtcctccgac gcaaggcgga
      421 gtactgtcct ccgacgcaag gtcggagtac tgtcctccga cactagaggt cggagtactg
      481 tcctccgcca tacgtagcag ggtagactgt agagggtata taatggaacc catcgcgtct
      541 cagcctcact ttgagctcct ccacacgaat tcacagccac catgctgtgc tgcatcagaa
      601 gaactaaacc ggttgagaag aatgaagagg ccgatcagga gctgcagtcg acggtaccgc
      661 gggcccggga tccaccggcc gccaccatgg tgagcaaggg cgaggaggac aacatggcca
      721 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt
      781 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
      841 aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt
      901 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
      961 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
     1021 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
     1081 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct
     1141 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc
     1201 tgaaggacgg cggccactac gacgccgagg tcaagaccac ctacaaggcc aagaagcccg
     1261 tgcagctgcc cggcgcctac aacgtcaaca tcaagctgga catcacctcc cacaacgagg
     1321 actacaccat cgtggaacag tacgagcgcg ccgagggccg ccactccacc ggcggcatgg
     1381 acgagctgta caagggaagc ggagctacta acttcagcct gctgaagcag gctggagacg
     1441 tggaggagaa ccccgggcct atgactattg ttcaagctgc ccaatcccgc tactccacca
     1501 aagcctttga tgcttcgcgc aaattgcctg aagagaaagt cgcggcagtg aaagagttaa
     1561 tccgcatgag tgcgtccagt gtcaactcgc aaccttggca ttttattgtc gcgagcagtg
     1621 aagagggaaa agcgcgcatc gccaaagcaa cacaaggtgg ttttgctgct aatgagcgca
     1681 agattttgga tgcttctcac gttgtggtgt tctgcgccaa aacggcgatt gatgaagcgt
     1741 acctactcga ccttttggaa agcgaagaca aagatggccg ctacgccgat gtcgaagcaa
     1801 aaaatggcat gcacgctggt cgttcatttt tcgtcaacat gcaccgcttt gacttgaaag
     1861 acgcgcatca ctggatggaa aagcaagttt acctcaatgt ggggacgctg ctattgggtg
     1921 cttctgcgat ggagatcgac gcggtgccaa ttgaaggctt cgatgccaag gtgcttgatg
     1981 aggagtttgg tctgcgtgag aagggcttta ccagcgtggt gattgtgccg ctgggttacc
     2041 atagcgaaga cgattttaat gctaagctgc caaaatcacg ttggtcagca gagactgttt
     2101 ttaccgaaat ctaagtcgag cacgaccgat cctgagaact tcagggtgag tttggggacc
     2161 cttgattgtt ctttcttttt cgctattgta aaattcatgt tatatggagg gggcaaagtt
     2221 ttcagggtgt tgtttagaat gggaagatgt cccttgtatc accatggacc ctcatgataa
     2281 ttttgtttct ttcactttct actctgttga caaccattgt ctcctcttat tttcttttca
     2341 ttttctgtaa ctttttcgtt aaactttagc ttgcatttgt aacgaatttt taaattcact
     2401 tttgtttatt tgtcagattg taagtacttt ctctaatcac ttttttttca aggcaatcag
     2461 ggtatattat attgtacttc agcacagttt tagagaacaa ttgttataat taaatgataa
     2521 ggtagaatat ttctgcatat aaattctggc tggcgtggaa atattcttat tggtagaaac
     2581 aactacaccc tggtcatcat cctgcctttc tctttatggt tacaatgata tacactgttt
     2641 gagatgagga taaaatactc tgagtccaaa ccgggcccct ctgctaacca tgttcatgcc
     2701 ttcttctctt tcctacagct cctgggcaac gtgctggttg ttgtgctgtc tcatcatttt
     2761 ggcaaagaat taattcctcg acgtccagac atgataagat acattgatga gtttggacaa
     2821 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     2881 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     2941 atgtttcagg ttcaggggga ggtgtgggag gttttttgtc gcgaatctag attaagattg
     3001 aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta cgttaagcat
     3061 gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat gattagagtc
     3121 ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa
     3181 ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat tgatcccccc tcgacagcaa
     3241 gcttataact tcgtatagca tacattatac gaagttattc aaccactcca ggcatagcta
     3301 gagccactga tgcatctata gagctgaagt aaagataggg ttgatgcaaa tcgatttaga
     3361 attttcaatg tagaaaaaac aaaataaaaa gtctattcac ttgatggata gattagtcaa
     3421 acaaacatag agtaggcact tcccttgact ctgtaaactt ttggatttaa cagattgtgc
     3481 actcctttta gttaaacaca ttagtccctg aaaactaaga ccctaaccat aatcccattg
     3541 tagacccacc tactcgggtg aatataaagc tcgcacagat catctcacag acatcagacc
     3601 ttggcaaagg agcatcctga acatctctac acactgaggc aaccctaggg ccgccaccat
     3661 gctgtgttgt atcagaagaa caaagcctgt ggagaagaat gaggaggctg accaggaact
     3721 gcagagcacg gtaccgcggt ccctgtatcc acctgtcgcc accatggtga gcaagggaga
     3781 ggagctgttc acaggagtgg tgcctatcct ggtggagctg gatggagatg tgaatggaca
     3841 caagttctct gtgagaggag agggagaggg agatgctaca aatggaaagc tgacactgaa
     3901 gttcatctgc acaacaggaa agctgcctgt gccttggcct acactggtga caacactgag
     3961 ccatggagtg cagtgttttg ctagataccc tgaccatatg aagcagcatg acttcttcaa
     4021 gtctgctatg cctgagggat atgtgcagga gagaacaatc ttcttcaagg atgatggaac
     4081 atacaagaca agagctgagg tgaagtttga gggagacaca ctggtgaaca gaattgaact
     4141 gaagggagtg gacttcaagg aggatggaaa catcctggga cacaagctgg agtacaactt
     4201 caacagccac aacatctaca ttatggctgt gaagcagaag aatggaatca aggtgaactt
     4261 caagatcaga cacaatgtgg aggatggatc tgtgcagctg gctgaccact accagcagaa
     4321 cacacctatt ggagatggac ctgtgctgct gcctgacagc cactacctga gcacacagtc
     4381 tgtgctgagc aaggacccta atgagaagag agaccatatg gtgctgctgg agttcagaac
     4441 agctgctgga atcacactgg gaatggacga gctgtacaag taaagcggcc gcgactctag
     4501 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     4561 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttatcga attctgcagg
     4621 aattgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac
     4681 tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt
     4741 gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa
     4801 gacaacctgt agggcacgag ggggggcccg aaacccgctg atcagcctcg actgtgcctt
     4861 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     4921 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     4981 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     5041 atagcaaggc atgctgggga tgcggtgggc tctatggtag ggataacagg gtaatataaa
     5101 ataaaataaa ataaaataaa ataaaataaa ataaaataaa ataaaataaa gcgcgcacta
     5161 gtgatatcaa gcttaaacaa gaatctctag ttttctttct tgcttttact tttacttcct
     5221 taatactcaa gtacaatttt aatggagtac ttttttactt ttactcaagt aagattctag
     5281 ccagatactt ttacttttaa ttgagtaaaa ttttccctaa gtacttgtac tttcacttga
     5341 gtaaaatttt tgagtacttt ttacacctct gctcgaccat atgggagagc tcccaacgcg
     5401 ttggatgcat agcttgagta ttctatagtg tcacctaaat agcttggcgt aatcatggtc
     5461 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5521 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5581 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5641 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5701 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5761 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5821 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5881 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5941 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6001 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6061 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6121 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6181 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6241 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6301 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6361 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6421 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6481 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6541 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6601 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6661 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6721 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6781 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6841 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6901 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6961 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7021 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7081 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7141 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7201 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     7261 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7321 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7381 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7441 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7501 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7561 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg atgcggtgtg
     7621 aaataccgca cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat
     7681 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     7741 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     7801 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     7861 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     7921 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     7981 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     8041 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     8101 gccgctacag ggcgcgtcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     8161 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     8221 agttgggtaa cgccagggtt ttcccagtca cgacgttgta
//
