LOCUS       Exported                8109 bp ds-DNA     circular SYN 21-FEB-2024
DEFINITION  Expresses ChrimsonR-tdTomato fusion after Flp-dependent 
            recombination.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-Ef1a-fDIO-ChrimsonR-tdTomato
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8109)
  AUTHORS   Sciolino NR, Hsiang M, Mazzone CM, Wilson LR, Plummer NW, 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     Natural locus coeruleus dynamics during feeding.
  JOURNAL   Sci Adv. 2022 Aug 19;8(33):eabn9134. doi: 10.1126/sciadv.abn9134. 
            Epub 2022 Aug 19.
  PUBMED    35984878
REFERENCE   2  (bases 1 to 8109)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 21, 2024 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8109
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1440..1473
                     /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(3025..4074)
                     /codon_start=1
                     /product="red light-drivable channelrhodopsin from 
                     Chlamydomonas noctigama (Klapoetke et al., 2014)"
                     /label=ChrimsonR
                     /note="K176R mutant with accelerated closing kinetics"
                     /translation="MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTE
                     WWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSI
                     AIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRY
                     FEWLLSCPVILIRLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIV
                     SCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLK
                     LSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEE
                     FVEEEDEDTV"
     regulatory      4071..4080
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(4094..4127)
                     /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    4236..4824
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4289..4309)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4707..4718)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4856..5332
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5055..5074)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5372..5512
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5372..5501
                     /label=AAV2 ITR
     rep_origin      5587..6042
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5674..5693)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5884..5905
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6059..6078)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6178..6200
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6238..6256)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6324..6428
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6429..7289
                     /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(6647..6666)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7460..8048
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7949..7968
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggag
     1441 aagttcctat tctctagaaa gtataggaac ttcgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag gaacttcggc
     1561 gcgcctgtag ctagctcatt acttatacag ctcatccatg ccgtacagaa acagatggtg
     1621 gcgtccttca gacctctcat actgttccac aatggtgtag tcttcattgt gggatgtgat
     1681 atccagcttg gtgtccacat agtaatagcc gggcagctgc actggtttct tggccatgta
     1741 gatggttttg aactccacca ggtagtggcc gccgtctttc agcttcaggg cctggtggat
     1801 ttcgcccttc agcactccgt ctctaggata cagccgttca gtgctggcct cccaccccat
     1861 tgttttcttc tgcatcacgg gcccatcggg tggaaaattg gtgccgcgca ttttgacttt
     1921 gtagatcaga gtgccatcct gcagagagga gtcctgtgtc acggtcacca ggcctccatc
     1981 ttcaaaattc atcactctct cccacttaaa gccctctggg aaagacagtt tcttgtaatc
     2041 aggaatgtca gcgggatgct tgacataggc cttacttccg tacataaact gaggagacag
     2101 gatatcccaa gcgaaaggca gtggtcctcc cttggtcact ttcagcttgg cagtctgtgt
     2161 gccctcgtaa ggcctccctt cgccctctcc ttcaatctca aattcgtgcc cattcatgct
     2221 gccctccatg cgcaccttaa acctcatgaa ctctttgatc acagccatat tgttatcctc
     2281 gcttgaggcg gttccactag accctgagcc ggtacttcca gtcccatggc ccaggaacag
     2341 atggtgtctc ccctcgctcc gttcatactg ctccacaata gtgtagtcct cgttgtgtga
     2401 agtgatatcc agttttgtgt ccacgtaata gtagcctggc agctgcacag gtttcttggc
     2461 catgtagatt gtcttgaact ccaccaggta atgtccccca tctttcagct tcagagcctg
     2521 gtggatttct cctttcagca ccccgtcgcg tggatacagc ctttcggtgg aggcctccca
     2581 tcccatagtt ttcttctgca tcacaggtcc atcaggggga aaatttgtcc ctctcatttt
     2641 caccttgtag atcagggtcc catcctgcag ggagctgtcc tgggtcacag tcaccaggcc
     2701 tccgtcctcg aagttcatca ctctttccca cttaaacccc tcggggaagc tcagtttctt
     2761 ataatcggga atgtcagctg gatgtttcac ataggccttg gagccgtaca taaactgtgg
     2821 gctcaggatg tcccaagcga atggcagagg cccgcccttt gtcactttca gcttggctgt
     2881 ctgggtgccc tcgtagggcc gtccctctcc ttctccctca atttcgaact cgtggccgtt
     2941 catgcttcct tccattctca ccttaaaccg catgaactct ttgatcactt cctcgccctt
     3001 actcactgct actaccggtg ccgccactgt gtcctcgtcc tcctcctcca cgaactcttc
     3061 cacttccacc tcctcgcctc cgatctccat cttggtggtc ttccggatgt cgccgtggat
     3121 caggatgtgc tcgtggatct tgatcctcag gtggtgggcc aggaaggtcc aaaactcctt
     3181 ggcgatgatg tcgcagatgc tgtggccgat gctgttggcg taagggctca gcttcagcag
     3241 tccttctggt cccactgccc agaggattgg gtagctgccc caagaggcga agtaagcgta
     3301 ggccatcagc ttcacgacca tgcggcaatg gcctttaggc acgctgtggt tggcttccac
     3361 gtagcacttg gcggcctgga agtacatgta gccgccgtag atgcaagaca cgatatacag
     3421 cagccacttg agccaatcgg tagccagtcc tgcggccatg ccgaacacga tcattcccac
     3481 gcaagacacg atcaggccca tggtccgctt gctgtagtcg ttcttcaggc cgctcaggtt
     3541 gctcagtctg atcaggatca cggggcaaga cagcagccac tcgaagtagc gcaggcaata
     3601 ggcgtggttg ccggtagaca ggtacactgt ggcggggctg ctgaactcct tgaagatctc
     3661 cagggtcacg aacagcacct cgacgcaaca gacgtagacc tcctcccaac cgcaagtggc
     3721 cttccaggcg ctgaagccgt agaatgtcag cagggcgatg gcgatgctga aagcaatcca
     3781 ctggcagacc tgggcgccga tcttttctcc tggtgtgcct ctgctgtgca gccaagcctg
     3841 cttcttctcc actcccacga ccaggtagcc gcctgtggcc tccacgaagc aatatccgta
     3901 tccggcgtcg ctcagtccgt aagaagggtc acaccaccac tcggtgctga agcactcgcc
     3961 tgtaggtgtc attcctccac agcccatgtc ctcgtggtgg taggggttag gccaagggtt
     4021 gatgcctccg gcggcaaaca gagatctggt ggcgctgctg atcagctcag ccatggtggc
     4081 ggcgcggcct agcgaagttc ctatactttc tagagaatag gaacttcttg ccttaaccca
     4141 gaaattatca ctgttattct ttagaatggt gcaaagagaa gttcctatac cttttgaaga
     4201 ataggaactt cgaattcgat atcaagctta tcgataatca acctctggat tacaaaattt
     4261 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     4321 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     4381 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     4441 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     4501 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     4561 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     4621 tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc
     4681 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     4741 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     4801 tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg
     4861 gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc
     4921 caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta
     4981 taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg
     5041 tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac
     5101 tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg
     5161 attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt
     5221 caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc
     5281 tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt
     5341 gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact
     5401 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     5461 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     5521 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     5581 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     5641 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     5701 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5761 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     5821 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5881 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     5941 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     6001 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     6061 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     6121 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     6181 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     6241 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     6301 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     6361 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     6421 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     6481 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     6541 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     6601 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     6661 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     6721 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     6781 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     6841 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     6901 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6961 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     7021 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     7081 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     7141 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     7201 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     7261 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     7321 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     7381 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     7441 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     7501 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     7561 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     7621 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     7681 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     7741 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     7801 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     7861 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     7921 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7981 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     8041 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     8101 ctcacatgt
//
