LOCUS       sequence-462659-        6607 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for tdTomato expression in Acanthamoeba via the
            Cre/loxP system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6607)
  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 6607)
  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: pUb-tdTomato-LP-Neo-LP
FEATURES             Location/Qualifiers
     source          1..6607
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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)."
     primer_bind     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             2714..4144
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLNGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     protein_bind    complement(4169..4202)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             4461..5255
                     /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(4515..4534)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5125..5144
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5319..5367
                     /note="synthetic polyadenylation signal"
     protein_bind    complement(5389..5422)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5966..5985)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5967..5985)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5992..6009)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5992..6008)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6001..6023)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6150..6605
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     6447..6468
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagcgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagctcga aattaaccct cactaaaggg aacttttgct gggtaccgcg taaaggcggg
     2221 ggtggtttac ttcaaggcaa gacttaaaca gagaacagaa cagagggggg taggaagcaa
     2281 agggcgacgt gatgtggaac gtacggtcat cggggagggt gtctttggcc cacgcctcgt
     2341 cgacgaggtc cagcagcagc gctgccttcg cgtgatgcac tcgcatgcag taacgcccgt
     2401 ccgcgctctt cccaaacaaa tacatgaaaa ttaaatacat acctacgcca aacccggtgc
     2461 aacatactac ttcgaaagga gccggcgtac catttcggaa ccaagagaga tgaacaatca
     2521 gctacgtcta acaatacaag gaaaatacaa tgaaatcatg ttttccttcc ggacaatcgg
     2581 tgtttgagct aaaaattctc caacaattcc gttctgcgaa cccaagggcc cagccgcata
     2641 aaaaggtgac ctgaccgcag cgtagaacac aaaacaaact acaacaagcg ctacacacaa
     2701 gccaacaaag cttatggtga gcaagggcga ggaggtcatc aaagagttca tgcgcttcaa
     2761 ggtgcgcatg gagggctcca tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     2821 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc
     2881 cttcgcctgg gacatcctgt ccccccagtt catgtacggc tccaaggcgt acgtgaagca
     2941 ccccgccgac atccccgatt acaagaagct gtccttcccc gagggcttca agtgggagcg
     3001 cgtgatgaac ttcgaggacg gcggtctggt gaccgtgacc caggactcct ccctgcagga
     3061 cggcacgctg atctacaagg tgaagatgcg cggcaccaac ttcccccccg acggccccgt
     3121 aatgcagaag aagaccatgg gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg
     3181 cgtgctgaat ggcgagatcc accaggccct gaagctgaag gacggcggcc actacctggt
     3241 ggagttcaag accatctaca tggccaagaa gcccgtgcaa ctgcccggct actactacgt
     3301 ggacaccaag ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     3361 gcgctccgag ggccgccacc acctgttcct ggggcatggc accggcagca ccggcagcgg
     3421 cagctccggc accgcctcct ccgaggacaa caacatggcc gtcatcaaag agttcatgcg
     3481 cttcaaggtg cgcatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg
     3541 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc
     3601 cctgcccttc gcctgggaca tcctgtcccc ccagttcatg tacggctcca aggcgtacgt
     3661 gaagcacccc gccgacatcc ccgattacaa gaagctgtcc ttccccgagg gcttcaagtg
     3721 ggagcgcgtg atgaacttcg aggacggcgg tctggtgacc gtgacccagg actcctccct
     3781 gcaggacggc acgctgatct acaaggtgaa gatgcgcggc accaacttcc cccccgacgg
     3841 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc accgagcgcc tgtacccccg
     3901 cgacggcgtg ctgaagggcg agatccacca ggccctgaag ctgaaggacg gcggccacta
     3961 cctggtggag ttcaagacca tctacatggc caagaagccc gtgcaactgc ccggctacta
     4021 ctacgtggac accaagctgg acatcacctc ccacaacgag gactacacca tcgtggaaca
     4081 gtacgagcgc tccgagggcc gccaccacct gttcctgtac ggcatggacg agctgtacaa
     4141 gtaacccggg ttaattaatt aaccatggat aacttcgtat agcatacatt atacgaagtt
     4201 atagatcctc tagaaaacga cgccttgcaa caagctgaga aaaaaccagg atcggcaagg
     4261 aaggattttc aacggaaacg ttggcatcac cgggtataaa aaggggccaa tttttttgtt
     4321 gatttgttgc gcgaattctt gctttcggca tcgaattcaa gggagaagga gtcgattcac
     4381 acatacaaca agatcctagg cttttgcaaa aagcttgatt cttctgacac aacagtctcg
     4441 aacttaaggc tagagccacc atgattgaac aagatggatt gcacgcaggt tctccggccg
     4501 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     4561 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     4621 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     4681 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     4741 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     4801 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     4861 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     4921 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     4981 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     5041 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     5101 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     5161 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     5221 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     5281 cgaccaagcg acgcccaacc tgccatcacg atggccgcaa taaaatatct ttattttcat
     5341 tacatctgtg tgttggtttt ttgtgtgaat cgatagcgat aagatatcat aacttcgtat
     5401 agcatacatt atacgaagtt atgagtgttg cctacagcta ctactttctg caaatacaac
     5461 acaactgttc ataaaccact acacgcagag tccacccgct tcgtgtgtga agggaatgtc
     5521 gcgcttttat tgcctgctct actcggcaag gaccttatca tcatcatttc gttgtggcct
     5581 tgcgttggtg gtgttcacgc ccttccattc agcacctccc tatcagggtt gctatcaatc
     5641 acgctactct acactcctcc tccttcatcc atcatcatca ccagtgtcgc ctcgtagcgt
     5701 gtcgcagtcc tcctcctgca agcaccagca gcaagcaggt cccttacgag gaggcttctt
     5761 ccatgtgcgc cttgagagcg ccattgccac ccttgtagag ctgagcgagc agcggcttaa
     5821 cgctgtcgcc agtgccgggc ttggggtcgc agtcgctggc ccaggtgaac ttgctcttgc
     5881 catctcccaa atcgtgcacc ttcatctgtt gagtagaagg tcactagttc tagagcggcc
     5941 gccaccgcgg tggagctcca attcgcccta tagtgagtcg tattacaatt cactggccgt
     6001 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     6061 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     6121 acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     6181 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     6241 tttcgcttcc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     6301 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     6361 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     6421 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     6481 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     6541 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     6601 aatttag
//
