LOCUS       sequence-462631-        7033 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cre recombinase in Acanthamoeba.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7033)
  AUTHORS   Aung JM, Joo SY, Na BK, Bang S, Shin M, Goo YK, Hong Y
  TITLE     Establishing a Cre/loxP-based genetic manipulation system for 
            Acanthamoeba: Targeted genome editing and stable reporter 
            expression.
  JOURNAL   Parasites Hosts Dis. 2025 Feb;63(1):25-36. doi: 10.3347/PHD.24078. 
            Epub 2025 Feb 25.
  PUBMED    40045678
REFERENCE   2  (bases 1 to 7033)
  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"; doi: "10.3347/PHD.24078";
            journalName: "Parasites Hosts Dis"; date: "2025-02"; volume: "63"; 
            issue: "1"; pages: "25-36"
COMMENT     Sequence Label: pGAPDH-Cre-HYG
FEATURES             Location/Qualifiers
     source          1..7033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             753..773
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             774..1799
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     CDS             join(1806..1808,1809..1811,1812..2522)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1806..1808,1809..1811,1812..2522)
                     /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(2112..2131)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2459..2480
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      2725..3180
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2812..2831)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3022..3043
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3297..3657
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3297..3488
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3320..3340)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     CDS             3828..4865
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPDRE
                     MGEAN"
     polyA_signal    4882..4930
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4971..4990)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5090..5112
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5150..5168)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5236..5340
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5341..5409,5410..6201)
                     /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(5559..5578)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6372..6960
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6861..6880
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atatgcaacc ctgtgacgag agacagggag tgctgttaga gaagagcgca cattcagcgc
       61 tcaagtggtc ttgcgtttgg tgtcagcacg aggtgcgcca gacccaggtc gcacgcaggc
      121 ccctcagcgc ggtcgccgtg tgttggtatt gtgtgcctga gtgatgcgga atgcgttctg
      181 cttgtaggcg tacgagaccg acgccaggcg gcctgtcgtg ctgggctcgt ccgcggcagg
      241 ctctgtaggg ctggcgcgga gtgggcgatc gtcctcccgc cgcgaggaaa cagcaacgga
      301 caggcttcat tgccacctca tcgccgcttt cgcgctcgag gccagggcgt ggcgagcagg
      361 taagaatctg agaagttgaa ttgtgaatta acacaaagaa gacgaacgaa cccgccgtgg
      421 tatggaagaa ggccacctca accaatctca agatcacctc ctaatcgtcc aacccggcct
      481 acaatcgcgc acatcagcac tcgatctcgc acttcggtcc gtttggccct cgagccaaag
      541 ccaagcagtt ttccaggcac cgttgaagtg cgaatagatg aaatttccgg tctaccccaa
      601 tgacatggcg ttttgtcaca acacatggcg ctgtgctggt ggtctcggtg gggcctattt
      661 atttggagtt gtttgcttgc tctctttcat ctttgtcttc ctacacagca gcaaacacca
      721 ctctctcact aagacatatg actagtatgg ccccaaagaa gaagcggaag gtctccaatt
      781 tactgaccgt acaccaaaat ttgcctgcat taccggtcga tgcaacgagt gatgaggttc
      841 gcaagaacct gatggacatg ttcagggatc gccaggcgtt ttctgagcat acctggaaaa
      901 tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac cggaaatggt
      961 ttcccgcaga acctgaagat gttcgcgatt atcttctata tcttcaggcg cgcggtctgg
     1021 cagtaaaaac tatccagcaa catttgggcc agctaaacat gcttcatcgt cggtccgggc
     1081 tgccacgacc aagtgacagc aatgctgttt cactggttat gcggcggatc cgaaaagaaa
     1141 acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact gatttcgacc
     1201 aggttcgttc actcatggaa aatagcgatc gctgccagga tatacgtaat ctggcatttc
     1261 tggggattgc ttataacacc ctgttacgta tagccgaaat tgccaggatc agggttaaag
     1321 atatctcacg tactgacggt gggagaatgt taatccatat tggcagaacg aaaacgctgg
     1381 ttagcaccgc aggtgtagag aaggcactta gcctgggggt aactaaactg gtcgagcgat
     1441 ggatttccgt ctctggtgta gctgatgatc cgaataacta cctgttttgc cgggtcagaa
     1501 aaaatggtgt tgccgcgcca tctgccacca gccagctatc aactcgcgcc ctggaaggga
     1561 tttttgaagc aactcatcga ttgatttacg gcgctaagga tgactctggt cagagatacc
     1621 tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg agatatggcc cgcgctggag
     1681 tttcaatacc ggagatcatg caagctggtg gctggaccaa tgtaaatatt gtcatgaact
     1741 atatccgtaa cctggatagt gaaacagggg caatggtgcg cctgctggaa gatggcgata
     1801 ctagtatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1861 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1921 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1981 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     2041 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2101 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     2161 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2221 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     2281 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     2341 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2401 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     2461 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2521 agtctagata agtcgaccca acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     2581 ggagatgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatccgata
     2641 aggatcgatc cgggctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     2701 ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2761 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2821 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2881 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2941 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3001 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3061 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3121 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3181 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgcggatc
     3241 tgcgcagcac catggcctga aataacctct gaaagaggaa cttggttagg taccttctga
     3301 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3361 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3421 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3481 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3541 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     3601 gagctattcc agaagtagtg aggaggcttt tttggaggat caaacgacgc cttgcaacaa
     3661 gctgagaaaa aaccaggatc ggcaaggaag gattttcaac ggaaacgttg gcatcaccgg
     3721 gtataaaaag gggccaattt ttttgttgat ttgttgcgcg aattcttgct ttcggcatcg
     3781 aattcaaggg agaaggagtc gattcacaca tacaacaaga tcctaggatg aaaaagcctg
     3841 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
     3901 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
     3961 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc
     4021 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gagttcagcg
     4081 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg
     4141 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg
     4201 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca
     4261 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg
     4321 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
     4381 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc
     4441 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
     4501 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc
     4561 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt
     4621 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
     4681 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg
     4741 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac
     4801 tcgccgatag tggaaaccga cgccccagca ctcgtccgga tcgggagatg ggggaggcta
     4861 actgaccatc acgatggccg caataaaata tctttatttt cattacatct gtgtgttggt
     4921 tttttgtgtg aatcgatagc gataaggatc cgcgtatggt gcactctcag tacaatctgc
     4981 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     5041 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     5101 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     5161 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     5221 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5281 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5341 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5401 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5461 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5521 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5581 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5641 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5701 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5761 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5821 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5881 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5941 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6001 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6061 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6121 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6181 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6241 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6301 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6361 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6421 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6481 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6541 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6601 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6661 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6721 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6781 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6841 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6901 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6961 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7021 gctcgacaga tct
//
