LOCUS       sequence-346604-        5071 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Leishmania cell free expression of zebrafish Cavin4b-EGFP. Parton 
            lab clone GRJ.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5071)
  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 5071)
  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-Cavin4b-EGFP
FEATURES             Location/Qualifiers
     source          1..5071
                     /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(1339..1363)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1365..1367,1368..2081)
                     /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(1365..1367,1368..2078)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1407..1428)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1668..1687)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2015..2036
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2083..2103)
                     /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(2111..2127)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2143..2277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2167..2186)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2221..2240
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2442..2462)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2442..2460)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2481..2497)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2481..2497)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2494..2516)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2505..2521
                     /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(2529..2535,2536..2553,2554..2559))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2574..2595
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2712..2729)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2883..3471)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2963..2982)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3642..4433,4434..4502))
                     /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     4265..4284
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4503..4607)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(4633..5071,1..17))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4770..4791)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4982..5001
                     /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 accatggctg ataaactggg actggcaggt gtggcggacg
      421 agcccagcgc catgaacatc ttctctctgc tggagagggt gtccgggatc atagacaatg
      481 ttcaggcctg ccagcagcgg atggaggaga ggcagctgga gctggagaac accgtcaaga
      541 ccatccaagc agatgtagtc aagctgacga aggagcacac ggcgaccagt ggcaccgtgg
      601 agaaacttct ggagaaaaca cgcaaggtca gctgccacat caaggacgtc cgcgtgcggg
      661 tggagaagca gaacgttcga gtcaagaagg tggaggaaac ccaagtcgag ctgctgaata
      721 agaacaagtt ccgtgtcgtc atctaccagg gagataatga ggtcccagct gtggctggtc
      781 ctaaagcctc tccgaaggga gcaatgggtg gagacgctgc tgcagaccca gatgcaccag
      841 aggctccgct ccctgactcg gatgaggaat acatggttgt tgaagaagcc gattcttcca
      901 ctgctgccaa actaaagaag accggcctga agcgcatcga aagcctgaag cagaccttct
      961 cacgcgagaa catgactaag accaaagaaa acctgggcac caaggtcaac aagctgggcg
     1021 agcgcatagt aacagccgaa cggcgtgaga aaatccgcca gtcaggagaa cgactgaaac
     1081 agtctggcga gcgcttcaag gaaaccatca caaaaaccgt accagcaaag ctgaatctga
     1141 agaaggaaag gacggtggct gagggtcagg agggggcaga agggaccaca gaaggaacag
     1201 cacccgtccc accaccgaag ggtcgtaaac ccagtcctga ggtggcctac acagagccaa
     1261 cgcagaaaca agaaggtcca gatgagggca agggtgagga atcagaagta ccgatgtatg
     1321 atatgaagca gctttctgac ccagctttct tgtacaaagt ggctgtgagc aagggcgagg
     1381 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1441 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     1501 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     1561 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1621 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     1681 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1741 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     1801 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     1861 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     1921 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     1981 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     2041 ccgccgggat cactctcggc atggacgagc tgtacaagta accaacttta ttatacatag
     2101 ttgataattc actggccgtc gttttacggt accatcgatg atgatccaga catgataaga
     2161 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     2221 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     2281 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     2341 agcaagtaaa acctctacaa atgtggtatg gctgattatg atctcgagat taccctgtta
     2401 tccctagcgt ggatcctagg actagtaccc agcttttgtt ccctttagtg agggttaatt
     2461 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     2521 attccacaca acatacgagc cgggagcata aagtgtaaag cctggggtgc ctaatgagtg
     2581 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     2641 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     2701 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2761 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2821 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2881 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2941 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3001 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3061 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     3121 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3181 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3241 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3301 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3361 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3421 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3481 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3541 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3601 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3661 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3721 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3781 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3841 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3901 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3961 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4021 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4081 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4141 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4201 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4261 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4321 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4381 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4441 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4501 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4561 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4621 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     4681 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     4741 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     4801 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     4861 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     4921 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     4981 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     5041 gcgtaaccac cacacccgcc gcgcttaatg c
//
