LOCUS       sequence-317822-        7393 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Flp-Dependent mCherry and Cre expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7393)
  AUTHORS   An JJ, Kinney CE, Tan JW, Liao GY, Kremer EJ, Xu B
  TITLE     TrkB-expressing paraventricular hypothalamic neurons suppress 
            appetite through multiple neurocircuits.
  JOURNAL   Nat Commun. 2020 Apr 7;11(1):1729. doi: 10.1038/s41467-020-15537-w.
  PUBMED    32265438
REFERENCE   2  (bases 1 to 7393)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2020-04-7"; volume: "11(1):1729. doi"; pages: " 
            10.1038/s41467-020-15537-w"
COMMENT     Sequence Label: pAAV-Ef1a-fDIO-mCherryP2ACre
FEATURES             Location/Qualifiers
     source          1..7393
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1210..1243
                     /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(1336..2367)
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             complement(2368..2424)
                     /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"
     CDS             complement(2434..3141)
                     /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(2709..2728)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2978..2996
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    complement(3148..3181)
                     /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    3290..3878
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3343..3363)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3761..3772)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3910..4386
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4109..4128)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4426..4566
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4426..4555
                     /label=AAV2 ITR
     rep_origin      4641..5096
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4728..4747)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4938..4959
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5113..5132)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5232..5254
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5292..5310)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5378..5482
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5483..5551,5552..6343)
                     /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(5701..5720)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6514..7102
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7003..7022
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7164..7293
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1201 gactccggag aagttcctat tctctagaaa gtataggaac ttcgcagaat ggtagctgga
     1261 ttgtagctgc tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag
     1321 gaacttcggc gcgccctaat cgccatcttc cagcaggcgc accattgccc ctgtttcact
     1381 atccaggtta cggatatagt tcatgacaat atttacattg gtccagccac cagcttgcat
     1441 gatctccggt attgaaactc cagcgcgggc catatctcgc gcggctccga cacgggcact
     1501 gtgtccagac caggccaggt atctctgacc agagtcatcc ttagcgccgt aaatcaatcg
     1561 atgagttgct tcaaaaatcc cttccagggc gcgagttgat agctggctgg tggcagatgg
     1621 cgcggcaaca ccattttttc tgacccggca aaacaggtag ttattcggat catcagctac
     1681 accagagacg gaaatccatc gctcgaccag tttagttacc cccaggctaa gtgccttctc
     1741 tacacctgcg gtgctaacca gcgttttcgt tctgccaata tggattaaca ttctcccacc
     1801 gtcagtacgt gagatatctt taaccctgat cctggcaatt tcggctatac gtaacagggt
     1861 gttataagca atccccagaa atgccagatt acgtatatcc tggcagcgat cgctattttc
     1921 catgagtgaa cgaacctggt cgaaatcagt gcgttcgaac gctagagcct gttttgcacg
     1981 ttcaccggca tcaacgtttt cttttcggat ccgccgcata accagtgaaa cagcattgct
     2041 gtcacttggt cgtggcagcc cggaccgacg atgaagcatg tttagctggc ccaaatgttg
     2101 ctggatagtt tttactgcca gaccgcgcgc ctgaagatat agaagataat cgcgaacatc
     2161 ttcaggttct gcgggaaacc atttccggtt attcaacttg caccatgccg cccacgaccg
     2221 gcaaacggac agaagcattt tccaggtatg ctcagaaaac gcctggcgat ccctgaacat
     2281 gtccatcagg ttcttgcgaa cctcatcact cgttgcatcg accggtaatg caggcaaatt
     2341 ttggtgtacg gtcagtaaat tggacatagg gccgggattc tcctccacgt cacctgcttg
     2401 tttgagtagt gagaagtttg ttgctccaga tcccttgtac agctcgtcca tgccgccggt
     2461 ggagtggcgg ccctcggcgc gttcgtactg ttccacgatg gtgtagtcct cgttgtggga
     2521 ggtgatgtcc aacttgatgt tgacgttgta ggcgccgggc agctgcacgg gcttcttggc
     2581 cttgtaggtg gtcttgacct cagcgtcgta gtggccgccg tccttcagct tcagcctctg
     2641 cttgatctcg cccttcaggg cgccgtcctc ggggtacatc cgctcggagg aggcctccca
     2701 gcccatggtc ttcttctgca ttacggggcc gtcggagggg aagttggtgc cgcgcagctt
     2761 caccttgtag atgaactcgc cgtcctgcag ggaggagtcc tgggtcacgg tcaccacgcc
     2821 gccgtcctcg aagttcatca cgcgctccca cttgaagccc tcggggaagg acagcttcaa
     2881 gtagtcgggg atgtcggcgg ggtgcttcac gtaggccttg gagccgtaca tgaactgagg
     2941 ggacaggatg tcccaggcga agggcagggg gccacccttg gtcaccttca gcttggcggt
     3001 ctgggtgccc tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt
     3061 cacggagccc tccatgtgca ccttgaagcg catgaactcc ttgatgatgg ccatgttatc
     3121 ctcctcgccc ttgctcacca tgctagcgaa gttcctatac tttctagaga ataggaactt
     3181 cttgccttaa cccagaaatt atcactgtta ttctttagaa tggtgcaaag agaagttcct
     3241 ataccttttg aagaatagga acttcgaatt cgatatcaag cttatcgata atcaacctct
     3301 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     3361 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     3421 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     3481 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     3541 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     3601 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     3661 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctatgttgc
     3721 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     3781 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     3841 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga
     3901 gagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt
     3961 gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac
     4021 taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
     4081 gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
     4141 caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct
     4201 cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg
     4261 tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc
     4321 tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct
     4381 gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga
     4441 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     4501 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     4561 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4621 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4681 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
     4741 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4801 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     4861 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     4921 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
     4981 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
     5041 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     5101 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     5161 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5221 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5281 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     5341 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5401 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5461 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5521 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5581 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5641 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5701 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5761 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5821 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5881 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     5941 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6001 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6061 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6121 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6181 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6241 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6301 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6361 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6421 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6481 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6541 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6601 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6661 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6721 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6781 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6841 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6901 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6961 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7021 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7081 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7141 ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc
     7201 cgcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag
     7261 agggagtggc caactccatc actaggggtt cctgcggccg cacgcgtaag ctttgcaaag
     7321 atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa
     7381 aggagtggga att
//
