LOCUS       sequence-310644-        4807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry clone containing postsynaptic HyCal component (HyNaC7)
            for HySyn system fused to eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4807)
  AUTHORS   Hawk JD, Wisdom EM, Sengupta T, Kashlan ZD, Colon-Ramos DA
  TITLE     A genetically encoded tool for reconstituting synthetic modulatory 
            neurotransmission and reconnect neural circuits in vivo.
  JOURNAL   Nat Commun. 2021 Aug 9;12(1):4795. doi: 10.1038/s41467-021-24690-9.
  PUBMED    34373460
REFERENCE   2  (bases 1 to 4807)
  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"; journalName: "Nat 
            Commun."; date: "2021-08-9"; volume: "12(1):4795. doi"; pages: " 
            10.1038/s41467-021-24690-9"
COMMENT     Sequence Label: pJH200_pE12_hyCal7-eGFP
FEATURES             Location/Qualifiers
     source          1..4807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(2225..2227,2228..2230,2231..2941)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     CDS             join(2225..2227,2228..2230,2231..2938)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(2270..2291)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2531..2550)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2878..2899
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3048..3067)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3049..3067)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3072..3088)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3072..3088)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3190..3209)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3201..4010
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3268..3287)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4157..4745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4646..4665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctta gctagcgcca ccatgagaat cagactgaag gagagaatga gaaaaattgt
      721 gcaggaaaga ctgaccgtga aacagatttt taaaagatat atcgagagct ctaccctgca
      781 cggattttgt tatgtgtgta tggacacatt cctgggaaga agactgattt gggccgtgct
      841 gatgatcctg ggagccatct atttcatttt taaactgaga tacggcatca aggaatactt
      901 tgactatcca ttcagcaccc tgtctacagt ggaatatgtg gacgatctgc tgtttccagc
      961 cgtgtctgtg tgcgctacca atagctacat tgcctctcag gtgtatacca atcagctgaa
     1021 tacaatgtat aaggagggaa gactgccact ggataacaac cagtctatcc cagaatataa
     1081 cattccagga gacgagctgg tgaagaccct gaagaacagc agcctgacaa ttgagtctct
     1141 gctgaagtat tgtgactgga tcatgcagga cacatctcac ccactggtga ccccaaataa
     1201 ttgtggagct ctgaatttta caagctattt taattacaag ggagagcagt gccataccct
     1261 gaacagcgga gctaaaggac atgaactgct gaaggtgagc gatgtgggaa tctctcatgg
     1321 atacgaactg gtgtttgacc tgcagacaaa cgaggttatt aagaactatc agctgagcgg
     1381 aatgagaatc gtgatccatg atcaggtgtt cccaccacag ctggtggatg gattctttat
     1441 cagcccagga ttcaagacct acatcaagct gggaattacc cagtctcagt ctctgccacc
     1501 accatattct acagaatgcg gacagaaaaa actgaagtat tatgctatct atagccagag
     1561 actgtgcctg ctggagaccc tgacagactt cacaggagac ctgtgcggat gtagagatgt
     1621 gtttatgcca gagaacggac tgccattctg tagcctgcag gaactgtata gctgcatgta
     1681 cccagctaaa gagtctttta gcgaattcac catgagaaaa gagtgtccaa gcgattgcga
     1741 agaaagaacc tttagctacg agctgtctga ggctagatat ctgcacaatc caccaattgg
     1801 actgtctctg agcagactgg ataaactgca gctggacaag tctctgccaa gcgaggctca
     1861 cctgtctaaa ctggctaaaa gcctgacacc aaaagaactg gatgcctaca tcgaaagcaa
     1921 catcatcagc gtgattctgt ttttcggaga taccagaatc gattaccacg aacaggaagc
     1981 cacaaacgac tttttccagt tcctgggaaa tatgggagga gaatttggac tgatgctggg
     2041 agcttccctg ctgaccttcg tggaattcat tgacctgttt attgtgctgc tgtaccacca
     2101 gatgctgaga ctgtataacc tgagaaagat tccagacatt tttgccagca aaagaaacag
     2161 aaataaggaa aaaaccaaga agtgtgagaa cgtgggtggc tcaggtggtg gcggttcagg
     2221 cggaatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     2281 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     2341 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     2401 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     2461 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     2521 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     2581 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     2641 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     2701 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     2761 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     2821 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     2881 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacta
     2941 gcttgtacaa agttggcatt ataagaaagc attgcttatc aatttgttgc aacgaacagg
     3001 tcactatcag tcaaaataaa atcattattt gccatccagc tgatatcccc tatagtgagt
     3061 cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa aatctctgat
     3121 gttacattgc acaagataaa ataatatcat catgaacaat aaaactgtct gcttacataa
     3181 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa
     3241 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     3301 caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac
     3361 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     3421 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     3481 tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt
     3541 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     3601 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     3661 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     3721 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
     3781 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     3841 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     3901 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     3961 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg
     4021 ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat
     4081 gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa
     4141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     4321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4801 acatgtt
//
