LOCUS       sequence-342358-        5401 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector expression of MmCav1-EGFP-APEX2 in Leishmania cell free 
            system. Parton lab clone JEJ.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5401)
  AUTHORS   Rae J, Ferguson C, Ariotti N, Webb RI, Cheng HH, Mead JL, Riches JD,
            Hunter DJ, Martel N, Baltos J, Christopoulos A, Bryce NS, Cagigas 
            ML, Fonseka S, Sayre ME, Hardeman EC, Gunning PW, Gambin Y, Hall TE,
            Parton RG
  TITLE     A robust method for particulate detection of a genetic tag for 3D 
            electron microscopy.
  JOURNAL   Elife. 2021 Apr 27;10. pii: 64630. doi: 10.7554/eLife.64630.
  PUBMED    33904409
REFERENCE   2  (bases 1 to 5401)
  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: "Elife."; 
            date: "2021-04-27"; volume: "10. pii: 64630. doi"; pages: " 
            10.7554/eLife.64630"
COMMENT     Sequence Label: SITS-MmCav1-EGFP-APEX2
FEATURES             Location/Qualifiers
     source          1..5401
                     /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"
     protein_bind    complement(919..943)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(945..947,948..950,951..1661)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(945..947,948..950,951..1661)
                     /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(990..1011)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1251..1270)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1598..1619
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1662..2408
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     protein_bind    complement(2413..2433)
                     /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(2441..2457)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2473..2607
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2497..2516)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2551..2570
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2772..2792)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2772..2790)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2811..2827)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2811..2827)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2824..2846)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2835..2851
                     /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(2859..2865,2866..2883,2884..2889))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2904..2925
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3042..3059)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3213..3801)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3293..3312)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3972..4763,4764..4832))
                     /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     4595..4614
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4833..4937)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(4963..5401,1..17))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5100..5121)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5312..5331
                     /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 accatgtctg ggggcaaata cgtagactcc gagggacatc
      421 tctacactgt tcccatccgg gaacagggca acatctacaa gcccaacaac aaggccatgg
      481 cagacgaggt gactgagaag caagtgtatg acgcgcacac caaggagatt gacctggtca
      541 accgcgaccc caagcatctc aacgacgacg tggtcaagat tgactttgaa gatgtgattg
      601 cagaaccaga agggacacac agtttcgacg gcatctggaa ggccagcttc accaccttca
      661 ctgtgacaaa atattggttt taccgcttgt tgtctacgat cttcggcatc ccaatggcac
      721 tcatctgggg catttacttt gccattctct ccttcctgca catctgggcg gttgtaccgt
      781 gcatcaagag cttcctgatt gagattcagt gcatcagccg cgtctactcc atctacgtcc
      841 ataccttctg cgatccactc tttgaagcta ttggcaagat attcagcaac atccgcatca
      901 gcacgcagaa agagatagac ccagctttct tgtacaaagt ggctatggtg agcaagggcg
      961 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1021 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     1081 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1141 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1201 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     1261 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1321 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1381 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     1441 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1501 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     1561 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1621 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggaaagtct tacccaactg
     1681 tgagtgctga ttaccaggac gccgttgaga aggcgaagaa gaagctcaga ggcttcatcg
     1741 ctgagaagag atgcgctcct ctaatgctcc gtttggcatt ccactctgct ggaacctttg
     1801 acaagggcac gaagaccggt ggacccttcg gaaccatcaa gcaccctgcc gaactggctc
     1861 acagcgctaa caacggtctt gacatcgctg ttaggctttt ggagccactc aaggcggagt
     1921 tccctatttt gagctacgcc gatttctacc agttggctgg cgttgttgcc gttgaggtca
     1981 cgggtggacc taaggttcca ttccaccctg gaagagagga caagcctgag ccaccaccag
     2041 agggtcgctt gcccgatccc actaagggtt ctgaccattt gagagatgtg tttggcaaag
     2101 ctatggggct tactgaccaa gatatcgttg ctctatctgg gggtcacact attggagctg
     2161 cacacaagga gcgttctgga tttgagggtc cctggacctc taatcctctt attttcgaca
     2221 actcatactt cacggagttg ttgagtggtg agaaggaagg tctccttcag ctaccttctg
     2281 acaaggctct tttgtctgac cctgtattcc gccctctcgt tgacaaatat gcagcggacg
     2341 aagatgcctt ctttgctgat tacgctgagg ctcaccaaaa gctttccgag cttgggtttg
     2401 ctgatgccta accaacttta ttatacatag ttgataattc actggccgtc gttttacggt
     2461 accatcgatg atgatccaga catgataaga tacattgatg agtttggaca aaccacaact
     2521 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     2581 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     2641 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg
     2701 gctgattatg atctcgagat taccctgtta tccctagcgt ggatcctagg actagtaccc
     2761 agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg
     2821 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cgggagcata
     2881 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     2941 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3001 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3061 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3121 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3181 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3241 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3301 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3361 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3421 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3481 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3541 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3601 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3661 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3721 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3781 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3841 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3901 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     3961 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4021 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4081 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4141 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4201 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4261 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4321 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4381 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4441 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4501 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4561 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4621 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4681 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4741 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4801 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4861 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     4921 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata
     4981 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5041 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5101 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5161 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5221 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5281 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5341 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5401 c
//
