LOCUS       sequence-435501-        6894 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent expression of Hm4Di fused to YFP; Chemogenetics.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6894)
  AUTHORS   Sanford CA, Soden ME, Baird MA, Miller SM, Schulkin J, Palmiter RD, 
            Clark M, Zweifel LS
  TITLE     A Central Amygdala CRF Circuit Facilitates Learning about Weak 
            Threats.
  JOURNAL   Neuron. 2017 Jan 4;93(1):164-178. doi: 10.1016/j.neuron.2016.11.034.
            Epub 2016 Dec 22.
  PUBMED    28017470
REFERENCE   2  (bases 1 to 6894)
  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.neuron.2016.11.034"; journalName: "Neuron"; date: 
            "2017-01-4- 4"; volume: "93"; issue: "1"; pages: "164-178"
COMMENT     Sequence Label: pAAV-FLEX-HM4Di-YFP
FEATURES             Location/Qualifiers
     source          1..6894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        220..1398
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          451..1389
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1346..1366
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1429..1462)
                     /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(1513..1546)
                     /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(1558..2271,2272..2274,2275..2277))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1603..1624)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1952..1971
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2211..2232
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2274..2283
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3733..3766
                     /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    3817..3850
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3874..3891)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3880..4104
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4146..4286
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4146..4275
                     /label=AAV2 ITR
     rep_origin      4361..4816
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4448..4467)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4658..4679
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4833..4852)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4952..4974
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5012..5030)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5098..5202
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5203..5271,5272..6063)
                     /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(5421..5440)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6234..6822
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6723..6742
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6884..6894,1..119)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc
       61 ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg
      121 cggccgcacg cgtaagcttt gcaaagatgg ataaagtttt aaacagagag gaatctttgc
      181 agctaatgga ccttctaggt cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg
      241 gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc
      301 ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc
      361 ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt
      421 tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct
      481 ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac
      541 gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct
      601 taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc
      661 gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
      721 taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg
      781 ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt
      841 gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga
      901 cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc
      961 gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg
     1021 ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
     1081 gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt
     1141 gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag cttttggagt
     1201 acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg
     1261 gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt
     1321 tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt
     1381 ccatttcagg tgtcgtgagg taccggatcc tctagagtcg actccggaat aacttcgtat
     1441 aggatacttt atacgaagtt atgcagaatg gtagctggat tgtagctgct attagcaata
     1501 tgaaacctct taataacttc gtatagcata cattatacga agttatggcg cgccctatta
     1561 cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac
     1621 catgtgatcg cgcttctcgt tggggtcttt gctcagggcg gactggtagc tcaggtagtg
     1681 gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc
     1741 gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg aagttcacct tgatgccgtt
     1801 cttctgcttg tcggccatga tatagacgtt gtggctgttg tagttgtact ccagcttgtg
     1861 ccccaggatg ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag
     1921 ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa
     1981 gatggtgcgc tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt
     2041 catgtggtcg gggtagcggg cgaagcactg caggccgtag ccgaaggtgg tcacgagggt
     2101 gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta
     2161 ggtggcatcg ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc
     2221 cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccatggt
     2281 ggcggccgcc ctggcagtgc cgatgttccg atactggcac agcagcaggt gccggaaggt
     2341 ctttttaaag gtggcgttgc acagagcata gcaggcaggg ttgatggtgc tgttgacgta
     2401 gcagagccag tagccaatgg accacaccgt gtcagggatg cagctctggc agaaggtgtt
     2461 caccaggacc atgacgttgt agggcgtcca ggtgaggatg aaggctagta gaatggcaaa
     2521 gatcgttcgt gtcactttgc gctcccgggc cgccatctgc cgcttcttgc gcacctggtt
     2581 gcgagcgatg ctggcgaact tgcgggccac gttggccgca gggcgcatgc cagccggcgt
     2641 ggcaggcaca atctcaatgg ctgtcacaca ctcattgcct gtctgcttcg tcacaatctg
     2701 gatcttggac catctggagg ctgggttgag ggcccgcggc tgcaggggag gggcgggcat
     2761 ggcgggcgtg gtggcctctg tggtggacag ctctgtggct gggcgttcct tggtgttctg
     2821 ggtggcactg cctgagctgg actcattgga agtgtcctta tcagccacgg ggcgcggtgg
     2881 cggtggcagc gctggcgggg gggcctcctc cagcttgcca ttgcgcagct cctcccgggc
     2941 ggcctccccg ggcgggggct tcttgacgct ctgcttcatt agtgggctct tgaggaaggc
     3001 cagcgtcttg gctttcttct ccttcgggcc ctcgggccgg tgcttgtgga ctcggctgcg
     3061 actggccagg gagatgtgga tgtacagcac cgtcatgatg accacaggca ggtagaagcc
     3121 agcaatggct gtgccaaagg tcactgctgg gttggacagg aactggatga agcactggtt
     3181 gtcgggcacc gtccgcttac ccaccacaaa ctgccagaac aagatggcag gcgcccagag
     3241 cacgaaggac agtacccagg cagcagcaat catgaggcct gccatcttgg tggtgcgccg
     3301 ggcagggtag gtgagaggct tggtgacgca gaagtagcgg tcaaagctga tgatgagaag
     3361 gttcatgacg gaggcgttgc tcaccacgca gtccagggcc agccacaggt cgcagaccac
     3421 ggcgcccagg ggccagtagc ccttgatgat gtacacggtg tagaggttca tggagaaggc
     3481 gcctatgatg agatcagcac acgccaggct gaagaggaag tagttgttga ctgtctgcag
     3541 ctgcctgttg accttgatgg acagcatcac caggatgttg cccacgacag tcaccaggct
     3601 cagggagcct gtcactgtgg caatgaagac catttccacc gtctcatagc gattgtggga
     3661 tgatgacgtg accaggcgca cggactgatt gcccgagctg ccattgacag gtgtgaagtt
     3721 ggccatgcta gcataacttc gtataaagta tcctatacga agttatttgc cttaacccag
     3781 aaattatcac tgttattctt tagaatggtg caaagaataa cttcgtataa tgtatgctat
     3841 acgaagttat gaattcgatg actcgctgat cagcctcgac tgtgccttct agttgccagc
     3901 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3961 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     4021 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     4081 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccaggtgc ggaccgagcg
     4141 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4201 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4261 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     4321 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     4381 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     4441 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4501 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4561 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4621 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4681 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     4741 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4801 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     4861 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4921 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4981 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5041 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5101 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5161 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5221 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5281 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5341 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5401 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5461 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5521 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5581 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5641 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     5701 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     5761 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     5821 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     5881 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     5941 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6001 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6061 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6121 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6181 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6241 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6301 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6361 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6421 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6481 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6541 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6601 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6661 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6721 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6781 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6841 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag gcag
//
