LOCUS       sequence-346505-        5104 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Leishmania cell free expression of zebrafish Cavin4a-EGFP. Parton 
            lab clone GRK.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5104)
  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 5104)
  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-Cavin4a-EGFP
FEATURES             Location/Qualifiers
     source          1..5104
                     /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"
     protein_bind    complement(1372..1396)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1398..1400,1401..2114)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1398..1400,1401..2111)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1440..1461)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1701..1720)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2048..2069
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2116..2136)
                     /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(2144..2160)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2176..2310
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2200..2219)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2254..2273
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2475..2495)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2475..2493)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2514..2530)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2514..2530)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2527..2549)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2538..2554
                     /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(2562..2568,2569..2586,2587..2592))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2607..2628
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2745..2762)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2916..3504)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2996..3015)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3675..4466,4467..4535))
                     /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     4298..4317
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4536..4640)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(4666..5104,1..17))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4803..4824)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5015..5034
                     /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 accatggaaa agagaggaga tgtcatatta ggggttgagg
      421 atgaatccgg gcagcccgtc tctgccctgt ccattctgtc tctgctggag cgtgtgtcaa
      481 caatcatcga tggtgtccag gcaagccaac agcgtatgga ggaacgccaa cagcagctcg
      541 agggctcggt cagcgctgtc cagtccgagc ttctcaaact ggctcgtgac cacggggcca
      601 cagcgacgac cgtcgacaaa ctgctgcaga aggcccggcg tgtgagcaca cacgttaaag
      661 aagtacgttc acgtgtggag aaacagaacg tgcgcgtgaa gaaagtggaa actacgcaag
      721 atgagctgct cacacgcaac aagttcaggg ttgtgatcta tcagggtgaa aaagaagtcc
      781 cctctgttgc tgtcaccaag actcccaagg gagcagggct tgctgagttg gaggtggagc
      841 cagatgagta tgacatccct gctgacctgt catctgatga agagtacatg gtggtggagg
      901 atgcggagtc gtccaggggc gcccgcctca aacagtccgg gttaaaaggc atcgagaaca
      961 tcaaggctgc gttctccaaa gagaacatga acaagacccg cgagaagacc cgagaaaacc
     1021 tcagcaagac caaggagagc ctgagtaaga ccggccagac gcttggaacc aaattcaaca
     1081 ctttgggtga gaagatcgtg ccacccgagc agcgagagaa gataaagcag agcagcgaga
     1141 ggctgaagga aaacatcgct aagaaagcgc ccaccaagga atccttcaaa atcaagctga
     1201 agaaggagcg cacggtcgct gaaggccagg agggtgcgga ggcggaacct gctgtgactc
     1261 cacccaaagg caggaagtcc agcccagatg tgacctacac agaggtggtt accgaaaaca
     1321 aaagggaggg tccagtatca gaggagggag caactcgtat ccatgaggac gacccagctt
     1381 tcttgtacaa agtggctgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     1441 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     1501 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     1561 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     1621 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     1681 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1741 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1801 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1861 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1921 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1981 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     2041 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     2101 agctgtacaa gtaaccaact ttattataca tagttgataa ttcactggcc gtcgttttac
     2161 ggtaccatcg atgatgatcc agacatgata agatacattg atgagtttgg acaaaccaca
     2221 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     2281 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     2341 caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     2401 atggctgatt atgatctcga gattaccctg ttatccctag cgtggatcct aggactagta
     2461 cccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     2521 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccgggagc
     2581 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     2641 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     2701 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     2761 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     2821 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     2881 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     2941 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3001 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3061 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3121 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3181 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     3241 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     3301 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     3361 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     3421 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     3481 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     3541 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     3601 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     3661 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     3721 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     3781 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     3841 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3901 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3961 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     4021 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4081 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4141 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4201 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     4261 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     4321 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     4381 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     4441 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     4501 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     4561 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     4621 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     4681 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     4741 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     4801 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     4861 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     4921 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     4981 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     5041 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     5101 atgc
//
