LOCUS       sequence-346509-        3906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite gateway middle entry clone containing zebrafish calcium 
            reporter GcAMP5. Parton lab clone ANR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3906)
  AUTHORS   Lo HP, Lim YW, Xiong Z, Martel N, Ferguson C, Ariotti N, Giacomotto 
            J, Rae J, Floetenmeyer M, Moradi SV, Gao Y, Tillu VA, Xia D, Wang H,
            Rahnama S, Nixon SJ, Bastiani M, Day RD, Smith KA, Palpant NJ, 
            Johnston WA, Alexandrov K, Collins BM, Hall TE, Parton RG
  TITLE     Cavin4 interacts with Bin1 to promote T-tubule formation and 
            stability in developing skeletal muscle.
  JOURNAL   J Cell Biol. 2021 Dec 6;220(12). pii: 212693. doi: 
            10.1083/jcb.201905065. Epub 2021 Oct 11.
  PUBMED    34633413
REFERENCE   2  (bases 1 to 3906)
  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: "J Cell 
            Biol."; date: "2021-12-6"; volume: "220(12). pii: 212693. doi"; 
            pages: " 10.1083/jcb.201905065. Epub 2021 Oct 11"
COMMENT     Sequence Label: pME-GcAMP5
FEATURES             Location/Qualifiers
     source          1..3906
                     /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"
     regulatory      672..681
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(678..686,687..704,705..707,708..740,741..743,744..767,
                     768..794,795..851,852..857,858..1127,1128..1145,1146..1580,
                     1581..1586,1587..2030)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             join(678..686,687..704,705..707,708..740,741..743,744..767,
                     768..794,795..851,852..857,858..1127,1128..1145,1146..1580,
                     1581..1586,1587..2030)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      774..783
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1064..1085
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1191..1212)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1452..1471)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(2031..2130)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2147..2166)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2148..2166)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2171..2187)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2171..2187)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2289..2308)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2300..3109
                     /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(2367..2386)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3256..3844
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3745..3764
                     /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 caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctgc cgccaccatg ggttctcatc atcatcatca tcatggtatg gctagcatga
      721 ctggtggaca gcaaatgggt cgggatctgt acgacgatga cgataaggat ctcgccacca
      781 tggtcgactc atcacgtcgt aagtggaata agacaggtca cgcagtcaga gctataggtc
      841 ggctgagctc actcgagaac gtctatatca aggccgacaa gcagaagaac ggcatcaagg
      901 cgaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc taccactacc
      961 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagcg
     1021 tgcagtccaa actttcgaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1081 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc ggtaccggag
     1141 ggagcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1201 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggtgag ggcgatgcca
     1261 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1321 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1381 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacatccag gagcgcacca
     1441 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1501 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1561 ggcacaagct ggagtacaac ctgccggacc aactgactga agagcagatc gcagaattta
     1621 aagaggcttt ctccctattt gacaaggacg gggatgggac aataacaacc aaggagctgg
     1681 ggacggtgat gcggtctctg gggcagaacc ccacagaagc agagctgcag gacatgatca
     1741 atgaagtaga tgccgacggt gacggcacaa tcgacttccc tgagttcctg acaatgatgg
     1801 caagaaaaat gaaatacaca gacagtgaag aagaaattag agaagcgttc cgtgtgtttg
     1861 ataaggatgg caatggctac atcagtgcag cagagcttcg ccacgtgatg acaaaccttg
     1921 gagagaagtt aacagatgaa gaggttgatg aaatgatcag ggaagcagac atcgatgggg
     1981 atggtcaggt aaactacgaa gagtttgtac aaatgatgac agcgaagtaa acccagcttt
     2041 cttgtacaaa gttggcatta taagaaagca ttgcttatca atttgttgca acgaacaggt
     2101 cactatcagt caaaataaaa tcattatttg ccatccagct gatatcccct atagtgagtc
     2161 gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg
     2221 ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa
     2281 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa
     2341 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     2401 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     2461 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     2521 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     2581 actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc
     2641 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     2701 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     2761 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     2821 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     2881 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     2941 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     3001 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     3061 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt
     3121 taattggttg taacactggc agagcattac gctgacttga cgggacggcg caagctcatg
     3181 accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa
     3241 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3301 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3361 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     3421 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3481 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3541 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3601 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3661 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3721 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3781 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3841 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3901 catgtt
//
