LOCUS       sequence-346602-        5562 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Leishmania cell free expression of zebrafish EGFP-Bin1a. Parton lab 
            clone GRQ.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5562)
  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 5562)
  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-Bin1a
FEATURES             Location/Qualifiers
     source          1..5562
                     /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(2574..2594)
                     /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(2602..2618)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2634..2768
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2658..2677)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2712..2731
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2933..2953)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2933..2951)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2972..2988)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2972..2988)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2985..3007)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2996..3012
                     /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(3020..3026,3027..3044,3045..3050))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3065..3086
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3203..3220)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3374..3962)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3454..3473)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4133..4924,4925..4993))
                     /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     4756..4775
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4994..5098)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5124..5562,1..17))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5261..5282)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5473..5492
                     /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 agtggtggct gaattaccga
     1141 tggggaaagg ggtgtccgct ggaaaaatag caagcaatgt acagaagaaa attactagag
     1201 cacaggagaa ggttatgcaa aagcttggaa aggcagatga gactaaagat ctggcgtttg
     1261 aagagggcgt gatcaacttt aataaacagt tggtggaagg taccaaactg cagaaagacc
     1321 ttcgggccta tttgactgct gtgaaagcca tgcacgagtc ttctagacgt ctgtacgagt
     1381 gcctggatga catgtatgag acagagtggt acggcagaaa gaaaatggac gcagttgtag
     1441 aggattcaga cctgctttgg acagacttcc atcagaaact cgtggatcat gcgcttatat
     1501 ccatggacac ttacctgggc cagtttccag acatcaaagc tcgcattgcg aaacgtgaca
     1561 gaaagctagt tgactttgac agcgcacggc acaactttgc tgctgtaaac aaaggcaaga
     1621 agaaagaagg ggtcaagatc accaagccag cctctttgct ggaaagggct gccccaagct
     1681 gggctcaggg gatcctgtcc gcccataatg tggctcaaac cagcctctcc aggagtcagg
     1741 cagaggagga gctggagagg gcacagaaag tctttgagga aattaacata gatttgcagg
     1801 aggaactgcc atctctgtgg aacagtcgtg ttggctttta tgtgaatacg ttccagagtg
     1861 tggcaggtct ggaggaaaag tttcacagag aaatgggaaa aattaatcag aatctttacg
     1921 atacactgga caaactggaa aatcaagata tgaccagtga aagaacacaa gatgccaatc
     1981 acactttaag cccaggagga ccaccagcca ttcccaaatc gccgtctaag ctaaggcctg
     2041 cagttcctcc tcctcccaaa gtaactccgt caaaggacct gaagcaggag agcatcatca
     2101 acctgtttga agaggccgcc actccaaaca tcagtgtcac ttcaccaaca gagtatgatg
     2161 ctccagccac tggtaatctt ctggatatgg atctggactc acttcaagct gccaccacac
     2221 cagctgcaca gacgtgggat tcatgggaga ctgcagaaac aacagctgtt acagaagagg
     2281 cagcagcagc tgaacctgta agtgagactg tgcaggagag cgccgagagc ccagagagcg
     2341 tagagatgcc agaatccttc ctgttcaaag tgaaagctat gcacgactac aatgccaccg
     2401 actctgatga gctggagtta aaggcgggcg acgtggtgct ggtagtgaat tatgaaaatc
     2461 cagacgatca ggatgacggc tggttgatgg gagtcaaaga ggctgattgg atccagcaga
     2521 aagatcttgg gaaaaaaggc gtgttccctg aaaacttcac ccagaggatg tgacaacttt
     2581 attatacata gttgataatt cactggccgt cgttttacgg taccatcgat gatgatccag
     2641 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     2701 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     2761 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     2821 aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat gatctcgaga
     2881 ttaccctgtt atccctagcg tggatcctag gactagtacc cagcttttgt tccctttagt
     2941 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3001 atccgctcac aattccacac aacatacgag ccgggagcat aaagtgtaaa gcctggggtg
     3061 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3121 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3181 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3241 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3301 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3361 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3421 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3481 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3541 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3601 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3661 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3721 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3781 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3841 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3901 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3961 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4021 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4081 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4141 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4201 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4261 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4321 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4381 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4441 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4501 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4561 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4621 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4681 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4741 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4801 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4861 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4921 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4981 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5041 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5101 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     5161 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     5221 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     5281 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     5341 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     5401 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     5461 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     5521 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gc
//
