LOCUS       sequence-342359-        4683 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector expression of MmCav1-APEX2 in Leishmania cell free system. 
            Parton lab clone IYW.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4683)
  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 4683)
  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-APEX2
FEATURES             Location/Qualifiers
     source          1..4683
                     /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             945..1691
                     /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(1695..1715)
                     /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(1723..1739)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1755..1889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1779..1798)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1833..1852
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2054..2074)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2054..2072)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2093..2109)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2093..2109)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2106..2128)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2117..2133
                     /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(2141..2147,2148..2165,2166..2171))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2186..2207
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2324..2341)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2495..3083)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2575..2594)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3254..4045,4046..4114))
                     /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     3877..3896
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4115..4219)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(4245..4683,1..17))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4382..4403)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4594..4613
                     /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 ggctggaaag tcttacccaa
      961 ctgtgagtgc tgattaccag gacgccgttg agaaggcgaa gaagaagctc agaggcttca
     1021 tcgctgagaa gagatgcgct cctctaatgc tccgtttggc attccactct gctggaacct
     1081 ttgacaaggg cacgaagacc ggtggaccct tcggaaccat caagcaccct gccgaactgg
     1141 ctcacagcgc taacaacggt cttgacatcg ctgttaggct tttggagcca ctcaaggcgg
     1201 agttccctat tttgagctac gccgatttct accagttggc tggcgttgtt gccgttgagg
     1261 tcacgggtgg acctaaggtt ccattccacc ctggaagaga ggacaagcct gagccaccac
     1321 cagagggtcg cttgcccgat cccactaagg gttctgacca tttgagagat gtgtttggca
     1381 aagctatggg gcttactgac caagatatcg ttgctctatc tgggggtcac actattggag
     1441 ctgcacacaa ggagcgttct ggatttgagg gtccctggac ctctaatcct cttattttcg
     1501 acaactcata cttcacggag ttgttgagtg gtgagaagga aggtctcctt cagctacctt
     1561 ctgacaaggc tcttttgtct gaccctgtat tccgccctct cgttgacaaa tatgcagcgg
     1621 acgaagatgc cttctttgct gattacgctg aggctcacca aaagctttcc gagcttgggt
     1681 ttgctgatgc ctaacaactt tattatacat agttgataat tcactggccg tcgttttacg
     1741 gtaccatcga tgatgatcca gacatgataa gatacattga tgagtttgga caaaccacaa
     1801 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     1861 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     1921 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
     1981 tggctgatta tgatctcgag attaccctgt tatccctagc gtggatccta ggactagtac
     2041 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
     2101 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccgggagca
     2161 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     2221 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     2281 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2341 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2401 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2461 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2521 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2581 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2641 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2701 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2761 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2821 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2881 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     2941 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     3001 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     3061 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3121 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3181 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3241 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3301 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3361 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     3421 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3481 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3541 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3601 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     3661 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     3721 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     3781 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     3841 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     3901 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3961 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     4021 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     4081 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4141 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4201 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa
     4261 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     4321 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     4381 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     4441 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     4501 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     4561 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     4621 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     4681 tgc
//
