LOCUS       sequence-461433-        7416 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Jonah-1 localizes to the outer cyst wall of Acanthamoeba tagged with
            eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7416)
  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 7416)
  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: Jonah-1
FEATURES             Location/Qualifiers
     source          1..7416
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(2286..2288,2289..3005)
                     /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(2328..2349)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2589..2608)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2936..2957
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3010..3131
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3047..3066)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3101..3120
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3317..3772
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3404..3423)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3614..3635
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3889..4249
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3889..4080
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3912..3932)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             4480..5274
                     /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(4534..4553)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5144..5163
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5338..5386
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5427..5446)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5546..5568
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5606..5624)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5692..5796
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5797..5865,5866..6657)
                     /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(6015..6034)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6828..7416
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7317..7336
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 gctcgacaca ccctcattgc aatttaaatg ccgtcttagg gggaggagca catgagggtg
      121 gcttcgatcc cgtggtgact gagtactaga ggctctgcga gcaacacctc ccagccccac
      181 gaaaaccaag gcctggtgcg tggtcagccg cgaaagaacg aaagcgggcg agcgttataa
      241 gaccaaaaaa cgtcagcgag atgaccaaat tgctagttca cccgaaagac gcgcccgcct
      301 gattggcgaa tgccgccaca ccagggtgcg ttgtgtctcc ttatgccaac tgagtgccca
      361 ccaaacgatc ccctactgct atgagtgctc taggcacatg gatccacgac gtgcaggaaa
      421 acttcgggat atgtcgcagc cacgaggtat ccggttcacg ctggtgtggg tgacattagg
      481 tcccacccaa ggtatataac atcctcgaca ctcctcctcc aaactcatca aagcgaactg
      541 ctcagctcac tcgaccagct ctctctccaa ccaactcgaa cactccatcc agcttggcct
      601 gctgtttctg ctttgtttcg actcatcgcc catctgcgat ctgaatgaag accacctacc
      661 tgctgctcgc tctcgccgcc ctgctcttct gcgctgtggt ggccgcctcc gccgacgacg
      721 atgaccgtcg cggctcggac atgaaccatt gggatgatga cgacgagggc cacgagtggg
      781 acaacgagtg ggaggacgag gaggatggcg agggccgcgg caggggtcag tgggacgacg
      841 gtcgcgagaa gtgtcgcacc accacgaaga agtgccgcac caccaccaag aagtgcggca
      901 ccaccaccaa gcactgcccc accaccacga ccaagcgctg cggcaccacg acgaaggatt
      961 gcggtacgac gaccaccaag tcctgcggta ccaccaccaa ggactgcggc accaccacca
     1021 tgaagccttg caccaccacc tcgaagcccg tcgagaagtg cgacttcgcc ggtcgcgaca
     1081 actgcctcaa ggcctcggag gacaagttcg aggagtgtgt cgaccgcaag aagtgcgcct
     1141 tcaccgtgtg cgagctcgag gcggaggaga agttcaagaa gtgcctccgt ggtcgcgacg
     1201 gccgcgacgg cggccgtgag aaccccttcg agcgcgaggg tgagaacgac aggcagggca
     1261 acgagaggtt cgatcagtgc aagaaggagc aactcgagga caagaagcag tgcctcatcc
     1321 agcgcaaggc ctcccacgcc ggctgcgtgg cggctaagga gtgcagggat ttcaagtgcc
     1381 tggccttcat caaggattgc aagccctgtc cgaccccggc ggccaccacc accaccgtga
     1441 gggtgaccac ccaccccccg accgtcggtt cctcgtgccc cgccgaggac ttcttcggcc
     1501 cggccgcgga cctgaacgtc ctcgtcaccg gttcggacaa gctgatctac gacctcgaca
     1561 tggtcaacgg cgacgtggag ggtcgcgtgg ccgtcaacgg cggcttccgc gtcaagagct
     1621 tcggcaccgc ccaagcctac tcgtgccccg ataccgccaa ctacgcctcg atgttcaacc
     1681 tcatcgtgaa cggcaagatg gattactcca acggccagct gttctgcggc tcgtccatca
     1741 gcctctcgag catgaacgac ccttccctgc gccagaagga tttcggcgcc accaacgccc
     1801 gcgtcgctgc cggcaagacc atcaaggatg tgacgggctt cgacttcgtg gccaccgccg
     1861 gctacctgca gggcgtgtcc aactacttct cgaagtacgc ccccggtacc cactcgcact
     1921 tcagggtgac cgaagtgtcc cccgccgacg tgaacaagct caacatcgag ggtggcgact
     1981 acgtgcaagt cattaacatc aagggctcgg gtgatatcaa cttcagcaac ttcgagatcc
     2041 ccaccgcgct caaccccagc caggtcatct acaacgtggt gggcaacaac aacatcaaga
     2101 tttcgggttt cggcctcaag ggtcacctcc tcgcgctcaa cagcgacgtc gtcctcgaga
     2161 acggccacgt ggccggcaac gtgtacgtgc gctccctcgt cggccagggc tccggccagg
     2221 tcaacctcgc tccttgcccg acctccggcg gtggctctgg aggtggtggg tccggcggtg
     2281 gctctgtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     2341 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     2401 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     2461 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     2521 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     2581 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2641 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2701 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     2761 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     2821 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2881 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     2941 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     3001 tctagaccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3061 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3121 aatgtatctt aaacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt
     3181 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatccga taaggatcga
     3241 tccgggctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     3301 cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3361 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3421 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3481 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3541 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3601 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     3661 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     3721 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttcctgatgc
     3781 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgcgga tctgcgcagc
     3841 accatggcct gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa
     3901 gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3961 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     4021 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     4081 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     4141 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     4201 ccagaagtag tgaggaggct tttttggagg atcaaacgac gccttgcaac aagctgagaa
     4261 aaaaccagga tcggcaagga aggattttca acggaaacgt tggcatcacc gggtataaaa
     4321 aggggccaat ttttttgttg atttgttgcg cgaattcttg ctttcggcat cgaattcaag
     4381 ggagaaggag tcgattcaca catacaacaa gatcctaggc ttttgcaaaa agcttgattc
     4441 ttctgacaca acagtctcga acttaaggct agagccacca tgattgaaca agatggattg
     4501 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     4561 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     4621 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     4681 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     4741 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     4801 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     4861 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     4921 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     4981 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     5041 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     5101 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     5161 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     5221 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     5281 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga tggccgcaat
     5341 aaaatatctt tattttcatt acatctgtgt gttggttttt tgtgtgaatc gatagcgata
     5401 aggatccgcg tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     5461 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     5521 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     5581 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     5641 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     5701 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5761 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5821 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5881 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5941 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     6001 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     6061 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     6121 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6181 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6241 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6301 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6361 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6421 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6481 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6541 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6601 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6661 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6721 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6781 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6841 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6901 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6961 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     7021 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     7081 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     7141 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     7201 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7261 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7321 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7381 ttgtgatgct cgtcaggggg gcggagccta tggaaa
//
