LOCUS       sequence-461219-        6291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Leo-A localizes to the inner cyst wall and ostioles of Acanthamoeba 
            tagged with eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6291)
  AUTHORS   Kanakapura Sundararaj B, Goyal M, Samuelson J
  TITLE     Cellulose binding and the timing of expression influence protein 
            targeting to the double-layered cyst wall of Acanthamoeba.
  JOURNAL   mSphere. 2024 Sep 25;9(9):e0046624. doi: 10.1128/msphere.00466-24. 
            Epub 2024 Aug 13.
  PUBMED    39136454
REFERENCE   2  (bases 1 to 6291)
  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: "mSphere."; 
            date: "2024-09-25"; volume: "9(9):e0046624. doi"; pages: " 
            10.1128/msphere.00466-24. Epub 2024 Aug 13"
COMMENT     Sequence Label: Leo-A
FEATURES             Location/Qualifiers
     source          1..6291
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      603..612
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1161..1163,1164..1880)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYKF"
     primer_bind     complement(1203..1224)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1464..1483)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1811..1832
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1885..2006
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1922..1941)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1976..1995
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2192..2647
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2279..2298)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2489..2510
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2764..3124
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2764..2955
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2787..2807)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             3355..4149
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3409..3428)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4019..4038
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4213..4261
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4302..4321)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4421..4443
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4481..4499)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4567..4671
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4672..4740,4741..5532)
                     /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     complement(4890..4909)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5703..6291
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6192..6211
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 gctcgacacg aaacggacag tcttgatcat ctctgtttat gtacagctct acataacatt
      121 tacatacaac atacatactc gacaaacaca tcagttgctt gttgtcttac attgttgttg
      181 ggcgtgtggc aggtcgacga tgagtggtgt acttagcatc atgtacataa cacagctaac
      241 cacaggtatt gaaccttcgc ccgccccact ttctacaatt catgttgtgg tggcgtcgac
      301 cagcggaact tcgctggtgt ggcggtggag gtgctgggct ggcgttcctg caccccgccc
      361 cccatggcga gcatctcgtg aacctttcca atcgcccacc aggcgcttta tcccaacaga
      421 gccctttctc tgattggcca gggctcacta cataaaccag agctcggcat cggcttttct
      481 tcacctcatc ttcaacactt gaacagcagc taacaacaag actacaagtc caacaccacc
      541 aacacaacac caacatgaac cgtttcgctc tctgcttctt cgtggtgctg tgtgcggcct
      601 cggccgccat gggccagagg tgtcctggct cggacttttt cccggaccag cgcggttgct
      661 gccccgtcgt ggtgaactcc gtgcctttct atgccgacgc ccagggttgc ttcccccgaa
      721 tcattagcgg agtcgccttc tacgctgacg ctggcggctg ctacccctcc tcggccggtg
      781 tgtaccgcag cgcgggcgcc gtctgccccg cggatcgcgt gtgcgagcct gagtgtcctg
      841 acatggccat cgtggtcgcc gaacagcagg gcggtgccag gctctgtgcc aacggcttct
      901 tctcggacgt caacggttgc tgcccgcgag tcctggtgga taatgtcccc tcgtatcgcg
      961 atgctcaagg ctgctaccct atctcgatcg cgtcggtgat cttctacgcc gacgtgggcc
     1021 gctgctaccc gaacagctcc ggtgtgtacc gcccggccgg tgccgtctgc cctgccggtc
     1081 aacagtgcgt cgtgagctgc gacaggaact acgccacgtc cggcggtggc tctggaggtg
     1141 gtgggtccgg cggtggctct gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     1201 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     1261 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     1321 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     1381 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     1441 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     1501 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     1561 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     1621 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     1681 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     1741 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     1801 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     1861 acgagctgta caagttctag acccaacttg tttattgcag cttataatgg ttacaaataa
     1921 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     1981 ttgtccaaac tcatcaatgt atcttaaaca acaacaattg cattcatttt atgtttcagg
     2041 ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa
     2101 tccgataagg atcgatccgg gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     2161 ccaacagttg cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg
     2221 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2281 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2341 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2401 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2461 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2521 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2581 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     2641 acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     2701 gcggatctgc gcagcaccat ggcctgaaat aacctctgaa agaggaactt ggttaggtac
     2761 cttctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     2821 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     2881 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2941 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3001 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3061 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggatcaa acgacgcctt
     3121 gcaacaagct gagaaaaaac caggatcggc aaggaaggat tttcaacgga aacgttggca
     3181 tcaccgggta taaaaagggg ccaatttttt tgttgatttg ttgcgcgaat tcttgctttc
     3241 ggcatcgaat tcaagggaga aggagtcgat tcacacatac aacaagatcc taggcttttg
     3301 caaaaagctt gattcttctg acacaacagt ctcgaactta aggctagagc caccatgatt
     3361 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     3421 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     3481 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     3541 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     3601 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     3661 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     3721 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     3781 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     3841 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     3901 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     3961 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4021 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4081 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4141 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     4201 cacgatggcc gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt
     4261 gaatcgatag cgataaggat ccgcgtatgg tgcactctca gtacaatctg ctctgatgcc
     4321 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4381 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4441 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     4501 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4561 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4621 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4681 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4741 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4801 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4861 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4921 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4981 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5041 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5101 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5161 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5221 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5281 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5341 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5401 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5461 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5521 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5581 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5641 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5701 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5761 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5821 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5881 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5941 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6001 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6061 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6121 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6181 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6241 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa a
//
