LOCUS       sequence-461438-        6506 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Luke-2 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 6506)
  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 6506)
  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: Luke-2
FEATURES             Location/Qualifiers
     source          1..6506
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1305..1307,1308..2024)
                     /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(1347..1368)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1608..1627)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1955..1976
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2029..2150
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2066..2085)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2120..2139
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2336..2791
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2423..2442)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2633..2654
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2908..3268
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2908..3099
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2931..2951)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             3499..4293
                     /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(3553..3572)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4163..4182
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4357..4405
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4446..4465)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4565..4587
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4625..4643)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4711..4815
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4816..4884,4885..5676)
                     /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(5034..5053)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5847..6435
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6336..6355
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctagggtcta acacgtgcca ttggctccca ttggtcggtg gcttacgtgt tcggccaatt
       61 gtgtttcagc actttttgtt cgaccaacga aattcacggc ttcaaactcg gtggtgcggt
      121 gatctgcttc cagtggtgtg cggggcgaca ccagcgcctt gagaccaagg gtgagccatt
      181 accgcggtgc agaactgttc acttatagct ccggttgcac tccacatggt ggtctgtgtc
      241 tatgggctta actgttgcgg gggtcgcaag atcccattgt tctgttggtg gtagagggcc
      301 aggggtgcaa ccagtgcact ttctctcgac tgggccacct cacctgtacc cgtcgaccac
      361 ctataaattg ttcaccactc gctcactcca ttcttcactc aaccaaccaa cacaccttgc
      421 gctaccaaca acctctacaa ccctcaacca tcatatgaat gaagcttctc ttctgcttga
      481 ccgttcttgc cgcccttatc ttctcctgtg cctctgctca gagttgcacg gcatcgctca
      541 accagacgcg ccaggcctcg tgggtggact ctgagcagtt cccgcgctcc ctgtggaccg
      601 tcgagatccg caacacgggc cagcaggccg tcaccaacgt gctcctctcc atccagggct
      661 ccatcaacca gatctgggag gtggtgctgg tcaacgacag cctctaccgc ctgccggact
      721 ggcgcctgca ggtgggcggc atcccggcgg gccagagcca cgtgttcggg ttcatcgtca
      781 acaacaacag cacggcggcg cccgtgtcgc tggtgagcgt ccagtgcggc aacgtcaccg
      841 tgccctcgcc ttcgccctcg gcctctgcct cgtcggtgcc ttcgccctcg gcctcggcct
      901 ctgctgcgcc cagccccagc gctggcgcga gtcccagctc gagcccgagg cccagcccgt
      961 cgggcgggtg ctcgctcctg gcgtcgcagg tggcacgctc aggcgccggc gggtcgtgga
     1021 gcgacaactc caatcggttc cagatctatg acatcacgct caacaacaat ggcgccagcc
     1081 gcttgaccca ggcctcgctc accatcgcca tcgccgccga ccaggccatc gcccagttct
     1141 ggaacctgga gcgggtcgac gccaccaaca cgttcaacat cgagagcttc ctgctgccgg
     1201 cgccgggcgg gtcgcagtcg ggcctgggct acgtgctgca gacgccgctc aactccacgt
     1261 ccgacgggtc gtccatcggc gccgtcaact tccgctgcac tagtgtgagc aagggcgagg
     1321 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1381 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     1441 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     1501 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1561 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     1621 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1681 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     1741 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     1801 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     1861 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     1921 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     1981 ccgccgggat cactctcggc atggacgagc tgtacaagtt ctagacccaa cttgtttatt
     2041 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     2101 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aacaacaaca
     2161 attgcattca ttttatgttt caggttcagg gggagatgtg ggaggttttt taaagcaagt
     2221 aaaacctcta caaatgtggt aaaatccgat aaggatcgat ccgggctggc gtaatagcga
     2281 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg
     2341 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2401 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2461 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2521 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2581 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2641 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2701 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2761 aattttaaca aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc
     2821 tgtgcggtat ttcacaccgc atacgcggat ctgcgcagca ccatggcctg aaataacctc
     2881 tgaaagagga acttggttag gtaccttctg aggcggaaag aaccagctgt ggaatgtgtg
     2941 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3001 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3061 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3121 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3181 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     3241 ttttggagga tcaaacgacg ccttgcaaca agctgagaaa aaaccaggat cggcaaggaa
     3301 ggattttcaa cggaaacgtt ggcatcaccg ggtataaaaa ggggccaatt tttttgttga
     3361 tttgttgcgc gaattcttgc tttcggcatc gaattcaagg gagaaggagt cgattcacac
     3421 atacaacaag atcctaggct tttgcaaaaa gcttgattct tctgacacaa cagtctcgaa
     3481 cttaaggcta gagccaccat gattgaacaa gatggattgc acgcaggttc tccggccgct
     3541 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     3601 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     3661 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     3721 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     3781 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     3841 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     3901 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     3961 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4021 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4081 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4141 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4201 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4261 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     4321 accaagcgac gcccaacctg ccatcacgat ggccgcaata aaatatcttt attttcatta
     4381 catctgtgtg ttggtttttt gtgtgaatcg atagcgataa ggatccgcgt atggtgcact
     4441 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4501 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4561 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     4621 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     4681 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4741 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4801 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4861 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4921 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     4981 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5041 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5101 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5161 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5221 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5281 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5341 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5401 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5461 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5521 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5581 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5641 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     5701 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5761 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5821 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5881 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5941 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6001 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6061 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6121 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6181 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6241 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6301 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6361 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6421 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6481 ccttttgctc acatggctcg acagat
//
