LOCUS       Exported                7054 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses tdTomato under control of eukaryotic Ef1a promoter after 
            Flp-dependent recombination.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-Ef1a-fDIO-tdTomato
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7054)
  AUTHORS   Sciolino NR, Mazzone CM, Plummer NW, Evsyukova I, Amin J, Smith KG, 
            McGee CA, Fry SA, Yang CX, Powell JM, Bruchas MR, Kravitz AV, 
            Cushman JD, Krashes MJ, Cui G, Jensen P
  TITLE     A role for the locus coeruleus in the modulation of feeding
  JOURNAL   bioRxiv 881599v1
REFERENCE   2  (bases 1 to 7054)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7054
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1450..1483
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(1595..3025)
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     regulatory      3022..3031
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(3039..3072)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    3181..3769
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3234..3254)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3652..3663)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3801..4277
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4000..4019)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4317..4457
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4317..4446
                     /label=AAV2 ITR
     rep_origin      4532..4987
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4619..4638)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4829..4850
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5004..5023)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5123..5145
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5183..5201)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5269..5373
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5374..6234
                     /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(5592..5611)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6405..6993
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6894..6913
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1441 gactccggag aagttcctat tctctagaaa gtataggaac ttcgcagaat ggtagctgga
     1501 ttgtagctgc tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag
     1561 gaacttcggc gcgcccggat caagcttcga attcttactt gtacagctcg tccatgccgt
     1621 acaggaacag gtggtggcgg ccctcggagc gctcgtactg ttccacgatg gtgtagtcct
     1681 cgttgtggga ggtgatgtcc agcttggtgt ccacgtagta gtagccgggc agttgcacgg
     1741 gcttcttggc catgtagatg gtcttgaact ccaccaggta gtggccgccg tccttcagct
     1801 tcagggcctg gtggatctcg cccttcagca cgccgtcgcg ggggtacagg cgctcggtgg
     1861 aggcctccca gcccatggtc ttcttctgca ttacggggcc gtcggggggg aagttggtgc
     1921 cgcgcatctt caccttgtag atcagcgtgc cgtcctgcag ggaggagtcc tgggtcacgg
     1981 tcaccagacc gccgtcctcg aagttcatca cgcgctccca cttgaagccc tcggggaagg
     2041 acagcttctt gtaatcgggg atgtcggcgg ggtgcttcac gtacgccttg gagccgtaca
     2101 tgaactgggg ggacaggatg tcccaggcga agggcagggg gccgcccttg gtcaccttca
     2161 gcttggcggt ctgggtgccc tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact
     2221 cgtggccgtt catggagccc tccatgcgca ccttgaagcg catgaactct ttgatgacgg
     2281 ccatgttgtt gtcctcggag gaggcggtgc cggagctgcc gctgccggtg ctgccggtgc
     2341 catgccccag gaacaggtgg tggcggccct cggagcgctc gtactgttcc acgatggtgt
     2401 agtcctcgtt gtgggaggtg atgtccagct tggtgtccac gtagtagtag ccgggcagtt
     2461 gcacgggctt cttggccatg tagatggtct tgaactccac caggtagtgg ccgccgtcct
     2521 tcagcttcag ggcctggtgg atctcgccct tcagcacgcc gtcgcggggg tacaggcgct
     2581 cggtggaggc ctcccagccc atggtcttct tctgcattac ggggccgtcg ggggggaagt
     2641 tggtgccgcg catcttcacc ttgtagatca gcgtgccgtc ctgcagggag gagtcctggg
     2701 tcacggtcac cagaccgccg tcctcgaagt tcatcacgcg ctcccacttg aagccctcgg
     2761 ggaaggacag cttcttgtaa tcggggatgt cggcggggtg cttcacgtac gccttggagc
     2821 cgtacatgaa ctggggggac aggatgtccc aggcgaaggg cagggggccg cccttggtca
     2881 ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc ctcgccctcg ccctcgatct
     2941 cgaactcgtg gccgttcatg gagccctcca tgcgcacctt gaagcgcatg aactctttga
     3001 tgacctcctc gcccttgctc accatggtgg cggctagcga agttcctata ctttctagag
     3061 aataggaact tcttgcctta acccagaaat tatcactgtt attctttaga atggtgcaaa
     3121 gagaagttcc tatacctttt gaagaatagg aacttcgaat tcgatatcaa gcttatcgat
     3181 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     3241 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     3301 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     3361 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     3421 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     3481 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     3541 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     3601 gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     3661 aatccagcgg accttcctcc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     3721 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccga
     3781 gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg
     3841 ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca
     3901 ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca
     3961 aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga
     4021 gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc
     4081 tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt
     4141 tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat
     4201 ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg
     4261 ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga
     4321 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     4381 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     4441 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4501 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     4561 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4621 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4681 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4741 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4801 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4861 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     4921 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     4981 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     5041 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     5101 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     5161 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     5221 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     5281 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5341 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5401 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5461 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5521 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5581 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5641 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5701 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5761 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5821 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5881 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5941 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6001 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6061 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6121 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6181 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6241 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6301 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6361 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6421 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6481 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6541 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6601 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6661 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6721 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6781 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6841 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6901 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6961 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7021 ttcctggcct tttgctggcc ttttgctcac atgt
//
