LOCUS       sequence-429065-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expresses hM4D and a green fluorescent label from a Cre-dependent
            virus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7302)
  AUTHORS   Atasoy D, Betley JN, Su HH, Sternson SM
  TITLE     Deconstruction of a neural circuit for hunger.
  JOURNAL   Nature. 2012 Aug 9;488(7410):172-7. doi: 10.1038/nature11270.
  PUBMED    22801496
REFERENCE   2  (bases 1 to 7302)
  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"; journalName: 
            "Nature"; date: "2012-08-9- 9"; volume: "488"; issue: "7410"; pages:
            "172-7"
COMMENT     Sequence Label: rAAV-CAG::FLEX-rev:: hM4D-2a-GFP
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   34..163
                     /label=AAV2 ITR
     promoter        474..754
                     /label=chicken beta-actin promoter
     primer_bind     1033..1055
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1117..1136
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1174..1207
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    1270..1303
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1373..2086,2087..2089))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1418..1439)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1767..1786
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2026..2047
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2090..2146)
                     /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"
     protein_bind    complement(3608..3641)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(3712..3745)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3779..4367
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3832..3852)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4250..4261)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4410..4531
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4421..4440)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4475..4494
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4693..4822
                     /label=AAV2 ITR
     rep_origin      complement(5059..5647)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5139..5158)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5818..6609,6610..6678))
                     /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     6441..6460
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6679..6783)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6809..7264)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6946..6967)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7158..7177
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgatcggtgc gggcctcttc gctattacgc cagctgcgcg ctcgctcgct cactgaggcc
       61 gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga
      121 gcgcgcagag agggagtggc caactccatc actaggggtt ccttgtagtt aatgattaac
      181 ccgccatgct acttatctac gtagccatgc tctaggaaga gtaccattga cgtcaataat
      241 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
      301 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
      361 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
      421 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt
      481 gagccccacg ttctgcttca ctctccccat ctcccccccc ctccccaccc ccaattttgt
      541 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggggcg
      601 cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc
      661 ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg
      721 gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc
      781 cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact
      841 cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta
      901 atgacggctt gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc
      961 tttgtgcggg gggagcggct cggggctgtc cgcgggggga cggctgcctt cgggggggac
     1021 ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc
     1081 atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt
     1141 ctcatcattt tggcaaagaa ttggatcatc cccataactt cgtataaagt atcctatacg
     1201 aagttatatc aaaataggaa gaccaatgct tcaccatcga cccgaattgc caagcatcac
     1261 catcgaccca taacttcgta taatgtatgc tatacgaagt tatactagcg gatccgaatt
     1321 cactagtctc gagcggccgc cagtgtgatg gatatctgca gaattcgccc ttttacttgt
     1381 acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt
     1441 gatcgcgctt ctcgttgggg tctttgctca gcttggactg ggtgctcagg tagtggttgt
     1501 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct
     1561 gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct
     1621 gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca
     1681 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt
     1741 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg
     1801 tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt
     1861 ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc
     1921 agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg
     1981 catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct
     2041 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacg gggccggggt
     2101 tctcctccac gtcgccggcc tgcttcagca gggagaagtt ggtggcgccg ctcccgtcga
     2161 ccctggcagt gccgatgttc cgatactggc acagcagcag gtgccggaag gtctttttaa
     2221 aggtggcgtt gcacagagca tagcaggcag ggttgatggt gctgttgacg tagcagagcc
     2281 agtagccaat ggaccacacc gtgtcaggga tgcagctctg gcagaaggtg ttcaccagga
     2341 ccatgacgtt gtagggcgtc caggtgagga tgaaggctag cagaatggca aagatcgttc
     2401 gtgtcacttt gcgctcccgg gccgccatct gccgcttctt gcgcacctgg ttgcgagcga
     2461 tgctggcgaa cttgcgggcc acgttggccg cagggcgcat gccagccggc gtggcaggca
     2521 caatctcaat ggctgtcaca cactcattgc ctgtctgctt cgtcacaatc tggatcttgg
     2581 accatctgga ggctgggttg agggcccgcg gctgcagggg aggggcgggc atggcgggcg
     2641 tggtggcctc tgtggtggac agctctgtgg ctgggcgttc cttggtgttc tgggtggcac
     2701 tgcctgagct ggactcattg gaagtgtcct tatcagccac ggggcgcggt ggcggtggca
     2761 gcgctggcgg gggggcctcc tccagcttgc cattgcgcag ctcctcccgg gcggcctccc
     2821 cgggcggggg cttcttgacg ctctgcttca ttagtgggct cttgaggaag gccagcgtct
     2881 tggctttctt ctccttcggg ccctcgggcc ggtgcttgtg gactcggctg cgactggcca
     2941 gggagatgtg gatgtacagc accgtcatga tgaccacagg caggtagaag ccagcaatgg
     3001 ctgtgccaaa ggtcactgct gggttggaca ggaactggat gaagcactgg ttgtcgggca
     3061 ccgtccgctt acccaccaca aactgccaga acaagatggc aggcgcccag agcacgaagg
     3121 acagtaccca ggcagcagca atcatgaggc ctgccatctt ggtggtgcgc cgggcagggt
     3181 aggtgagagg cttggtgacg cagaagtagc ggtcaaagct gatgatgaga aggttcatga
     3241 cggaggcgtt gctcaccacg cagtccaggg ccagccacag gtcgcagacc acggcgccca
     3301 ggggccagta gcccttgatg atgtacacgg tgtagaggtt catggagaag gcgcctatga
     3361 tgagatcagc acacgccagg ctgaagagga agtagttgtt gactgtctgc agctgcctgt
     3421 tgaccttgat ggacagcatc accaggatgt tgcccacgac agtcaccagg ctcagggagc
     3481 ctgtcactgt ggcaatgaag accatttcca ccgtctcata gcgattgtgg gatgatgacg
     3541 tgaccaggcg cacggactga ttgcccgagc tgccattgac aggtgtgaag ttggccatgg
     3601 tactagtata acttcgtata ggatacttta tacgaagtta tcattgggat tcttcctatt
     3661 ttgatccaag catcaccatc gaccctctag tccagatctc accatcgacc cataacttcg
     3721 tatagcatac attatacgaa gttatgtccc tcgaagaggt tcgaattagc ttatcgataa
     3781 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     3841 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     3901 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     3961 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     4021 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     4081 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     4141 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     4201 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     4261 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     4321 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg
     4381 acccgggcgg ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac
     4441 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4501 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     4561 tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     4621 taaaatcgat aaggatcttc ctagagcatg gctacgtaga taagtagcat ggcgggttaa
     4681 tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     4741 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     4801 cagtgagcga gcgagcgcgc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4861 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4921 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4981 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5041 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5101 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5161 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5221 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5281 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5341 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5401 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5461 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5521 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5581 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5641 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5701 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5761 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5821 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5881 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5941 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6001 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6061 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6121 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6181 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6241 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6301 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6361 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6421 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6481 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6541 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6601 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6661 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6721 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6781 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     6841 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     6901 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     6961 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     7021 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     7081 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     7141 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     7201 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     7261 gcgtcccatt cgccattcag gctgcgcaac tgttgggaag gg
//
