LOCUS       sequence-442271-        5897 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GCaMP6f under the rat pro-melanin-concentrating hormone 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5897)
  AUTHORS   Guo R, Wang Y, Yan R, Chen B, Ding W, Gorczyca MT, Ozsoy S, Cai L, 
            Hines RL, Tseng GC, Allocca G, Dong Y, Fang J, Huang YH
  TITLE     Rapid Eye Movement Sleep Engages Melanin-Concentrating Hormone 
            Neurons to Reduce Cocaine Seeking.
  JOURNAL   Biol Psychiatry. 2022 Dec 1;92(11):880-894. doi: 
            10.1016/j.biopsych.2022.06.006. Epub 2022 Jun 13.
  PUBMED    35953320
REFERENCE   2  (bases 1 to 5897)
  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: 
            "10.1016/j.biopsych.2022.06.006"; journalName: "Biol Psychiatry"; 
            date: "2022-12-1- 1"; volume: "92"; issue: "11"; pages: "880-894"
COMMENT     Sequence Label: pAAV-MCHpr-GCaMP6f
FEATURES             Location/Qualifiers
     source          1..5897
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      626..635
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(632..640,641..658,659..661,662..694,695..697,698..721,
                     722..748,749..805,806..811,812..1081,1082..1099,1100..1534,
                     1535..1540,1541..1984)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      728..737
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1018..1039
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1145..1166)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1406..1425)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2024..2612
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2077..2097)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2495..2506)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2644..3120
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2843..2862)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3160..3300
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3160..3289
                     /label=AAV2 ITR
     rep_origin      3375..3830
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3462..3481)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3672..3693
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3847..3866)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3966..3988
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4026..4044)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4112..4216
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4217..4285,4286..5077)
                     /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(4435..4454)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5248..5836
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5737..5756
                     /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 gcgttctaga gataacttct atttaataag gattaaaagg
      181 gcataaagct tttatcctta ttatccatga attaaataac acgtcttaat atcttaatcg
      241 gaaagtacat agaaggtaac tgtaatattg ggagagcctg gtgtcctatg gtcttcctta
      301 ataacatccc actttcaaga ggctataatc tactcacaga aagccttctc tcaaagacct
      361 taatcagatt ctgggaaaat tcacacacga aataaacaac aggccactaa atccaaaaca
      421 atgcaagcat tagcagcttt acaaaggcga ggctctacgg ggtgattcat ttctaaaaag
      481 aaaagataag gccttcaagt gttttctatt caggcacaaa gtatataaag gtaggaatca
      541 ttcagtcgcc agcagagtgt ggcattctcc ccacattctc ttcggcttta cggagcagca
      601 aacagggtcg actctagagg atcccgccac catgggttct catcatcatc atcatcatgg
      661 tatggctagc atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa
      721 ggatctcgcc accatggtcg actcatcacg tcgtaagtgg aataagacag gtcacgcagt
      781 cagagctata ggtcggctga gctcactcga gaacgtctat atcaaggccg acaagcagaa
      841 gaacggcatc aaggcgaact tcaagatccg ccacaacatc gaggacggcg gcgtgcagct
      901 cgcctaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
      961 ccactacctg agcgtgcagt ccaaactttc gaaagacccc aacgagaagc gcgatcacat
     1021 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1081 gggcggtacc ggagggagca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1141 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1201 tgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1261 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg
     1321 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacat
     1381 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1441 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1501 cggcaacatc ctggggcaca agctggagta caacctgccg gaccaactga ctgaagagca
     1561 gatcgcagaa tttaaagagg aattctccct atttgacaag gacggggatg ggacaataac
     1621 aaccaaggag ctggggacgg tgatgcggtc tctggggcag aaccccacag aagcagagct
     1681 gcaggacatg atcaatgaag tagatgccga cggtgacggc acaatcgact tccctgagtt
     1741 cctgacaatg atggcaagaa aaatgaaata cagggacacg gaagaagaaa ttagagaagc
     1801 gttcggtgtg tttgataagg atggcaatgg ctacatcagt gcagcagagc ttcgccacgt
     1861 gatgacaaac cttggagaga agttaacaga tgaagaggtt gatgaaatga tcagggaagc
     1921 agacatcgat ggggatggtc aggtaaacta cgaagagttt gtacaaatga tgacagcgaa
     1981 gtaacgcgtg tacaagtaag aattcgatat caagcttatc gataatcaac ctctggatta
     2041 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     2101 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     2161 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     2221 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     2281 cacctgtcag ctcctttccg gaactttcgc tttccccctc cctattgcca cggcggaact
     2341 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     2401 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg
     2461 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     2521 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     2581 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc
     2641 tacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact
     2701 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg
     2761 tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag
     2821 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct
     2881 tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag
     2941 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga
     3001 cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca
     3061 ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt
     3121 ctgattttgt aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt
     3181 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     3241 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     3301 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     3361 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3421 gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
     3481 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3541 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     3601 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3661 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta
     3721 ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
     3781 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     3841 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     3901 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3961 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     4021 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     4081 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4141 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4201 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4261 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     4321 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4381 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4441 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4501 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     4561 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     4621 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     4681 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     4741 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     4801 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     4861 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     4921 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     4981 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5041 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5101 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5161 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5221 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5281 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5341 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5401 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5461 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5521 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5581 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5641 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5701 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5761 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5821 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5881 gccttttgct cacatgt
//
