LOCUS       sequence-346525-        5208 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Leishmania cell free expression of zebrafish EGFP-Bin1b. Parton lab 
            clone GRS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5208)
  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 5208)
  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: SITS-EGFP-Bin1b
FEATURES             Location/Qualifiers
     source          1..5208
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    117..137
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     168..187
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        168..186
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             330..353
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     protein_bind    353..377
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      378..387
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(384..386,387..389,390..1100)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(384..386,387..389,390..1100)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(429..450)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(690..709)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1037..1058
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1102..1126)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2220..2240)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(2248..2264)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2280..2414
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2304..2323)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2358..2377
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2579..2599)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2579..2597)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2618..2634)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2618..2634)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2631..2653)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2642..2658
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2666..2672,2673..2690,2691..2696))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2711..2732
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2849..2866)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3020..3608)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3100..3119)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3779..4570,4571..4639))
                     /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     4402..4421
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4640..4744)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(4770..5208,1..17))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4907..4928)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5119..5138
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
       61 ggtgcgggcc tcttcgctat tacgccagtc gactagggat aacagggtaa tgaattcaac
      121 tttgtataga aaagttgaca tttccccgaa aagtgccacc tgacgtctaa tacgactcac
      181 tatagggaca tcttaagttt attttatttt attttatttt attttatttt attttatttt
      241 attttatttt attttattta accatgacag taatgtataa agtctgtaaa gacattaaac
      301 acgtaagtga aaccatgggc acctctggtc accaccatca ccaccatcac caccaagttt
      361 gtacaaaaaa gcaggctgcc accatggtga gcaagggcga ggagctgttc accggggtgg
      421 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
      481 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
      541 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
      601 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
      661 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
      721 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
      781 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
      841 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
      901 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
      961 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     1021 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     1081 gcatggacga gctgtacaag cacccagctt tcttgtacaa agtggtggca gaggttggga
     1141 aaggagtcaa cgccgggaaa ctggccagta atgtacagaa gaggttaacc cgagcacaag
     1201 agaaggtgat gcagaaactg ggaaaggcgg atgaaaccaa agacacagcc tttgaggagc
     1261 aagtagccaa ctttaacaaa cagatgaatg agggcaccaa actgcagaaa gacttgaaag
     1321 cgtatttgct ggcagtcaaa acaatgcacg agtgttccaa gcgtctgcac gactgtctgg
     1381 cagaaatgta tgaccctgag tggtttggca aagaggaggt ggactccatc gcagaggaca
     1441 ctgacctcct gtggcaagat ttccacgaga agcttttgga caatgctgta atttccatgg
     1501 acgcatactt ggctcagttt ccagatataa aggctcgcat tgcaaagcgt gaaaggaagc
     1561 tggtggactt tgacagtgcc agacatcatt ttgcctctgt acaaaaaggc aagaaaaagg
     1621 acgaggccaa aattgccaag gctgaggaac acttgggcag agctcagaaa gtgtttgagg
     1681 agatcaatta tgatctccag gaggaacttc ccaccctgtg gaacagtcgt attggcctct
     1741 atgttaacac attccggagt gtgtctggcc tggaggaaac atttcacagg gacatgggca
     1801 aactgaacca gaatctgagt gatattatga ccaaactgga agagcagcgt gttgccaaaa
     1861 aaggtgtcac gacagcaagc actgggaaga gtgacgatgc agcaaaccac aatcaggaga
     1921 cgtcagatgc acctaaatct ccatcaaaga atgaagcccc aacggtgaca aatggctcct
     1981 cagatacaga gcttcctcca tctgttctct ttaaggtgaa aacaatgcac gactacagtg
     2041 caacagacag tgatgaactt gacctgaagg ctggtgacat tgtgcttgtg cttccctttg
     2101 ccaacccaga tgaagcggat gatggctggt taatgggtgt aaaagagtcc cactggcttc
     2161 agaacaaaaa cctcctagca aaaggagtct tccctgaaaa cttcacccag aaactgtgac
     2221 aactttatta tacatagttg ataattcact ggccgtcgtt ttacggtacc atcgatgatg
     2281 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     2341 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     2401 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     2461 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc
     2521 tcgagattac cctgttatcc ctagcgtgga tcctaggact agtacccagc ttttgttccc
     2581 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2641 attgttatcc gctcacaatt ccacacaaca tacgagccgg gagcataaag tgtaaagcct
     2701 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2761 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2821 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2881 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2941 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3001 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3061 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3121 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3181 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3241 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3301 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3361 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3421 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3481 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3541 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3601 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3661 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3721 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3781 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3841 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3901 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3961 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4021 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4081 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4141 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4201 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4261 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4321 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4381 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4441 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4501 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4561 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4621 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4681 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4741 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
     4801 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     4861 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     4921 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     4981 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     5041 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     5101 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     5161 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgc
//
