LOCUS       sequence-259421-        8357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mCherry protein and a fusion protein of Nluc-neo inserting
            into Cryptosporidium parvum UPRT locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8357)
  AUTHORS   Wilke G, Funkhouser-Jones LJ, Wang Y, Ravindran S, Wang Q, Beatty 
            WL, Baldridge MT, VanDussen KL, Shen B, Kuhlenschmidt MS, 
            Kuhlenschmidt TB, Witola WH, Stappenbeck TS, Sibley LD
  TITLE     A Stem-Cell-Derived Platform Enables Complete Cryptosporidium 
            Development In Vitro and Genetic Tractability.
  JOURNAL   Cell Host Microbe. 2019 Jun 18. pii: S1931-3128(19)30252-5. doi: 
            10.1016/j.chom.2019.05.007.
  PUBMED    31231046
REFERENCE   2  (bases 1 to 8357)
  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: "Cell Host 
            Microbe. 2019 Jun 18. pii: S1931-3128(19)30252-5. doi: 
            10.1016/j.chom.2019.05.007."
COMMENT     Sequence Label: UPRT-mCh-Nluc-P2A-neo-UPRT
FEATURES             Location/Qualifiers
     source          1..8357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4145..4163)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4413..4432
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             5638..6150
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             6166..6222
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(7963..7980)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7963..7979)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7972..7994)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(8188..8207)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8307..8329
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctt gcatgcctgc agagttataa gcaccaagct aaacaaatat
     2281 taaaaagcat cgggaatgat caaggagtag aaaaatgttc tattgatagt ggaaattatg
     2341 tcttccacta cattgtaaat gatggtatta tttatatgac tttatcggga aagtcatacc
     2401 cgaagaagtt ggctttttct tttctagatg aaataaaaag gttatttatt gaagaactag
     2461 ggagagaatt tgcaaataga gatgaattca agagcctgat tgatgcaata gataagccgt
     2521 attatttcat aaagtttgac cgaataattc aacgatgcaa atccgaatat aaggacccaa
     2581 actcgaacaa gtcgctacaa aaactaaatg actcgctaat tgaggtatgt ttactacttg
     2641 tttttaacca taatgctcaa aaaaattaac aattcaggta actaatataa tgagaagaaa
     2701 tgtggatgat attttactga gaggtgaaaa tcttaatgat gttgaaagaa aagccaatga
     2761 tttaaaattt gcatccctgg aattttctag agctgcaaaa aagctttcat tgcatgcatt
     2821 aattcaaaaa tatgcaccaa tattggcctt atcggctata gttttgatca tcattatatg
     2881 gaagcttatt ttataaattc aaatttctaa attgcggaat aaaaaaaact ttgaacatta
     2941 gcgccataac tttttttttg aaaaaatttt tgcaagtatt acgtttaaaa gtgctttata
     3001 aattttactt aaaatcaata gtaaactcaa ttgagagctt tcaatcagct cagaatgagt
     3061 tggttataaa cagtaataat agaaagcgtt agggctataa aaatgaccaa ataagtcatt
     3121 tagcatcata agtatataca ttagatacag aatgagttca aaataaataa aaaattagac
     3181 ttacttacaa attctaagct atagtataaa agaactaaga agatttgatg aatttgctat
     3241 accattttaa atatttgttt atagtttttt gatattaata taaggactat taagtgaaaa
     3301 ggaaactaca catatttccg ccaattcatt gtattgcatc gcacacataa atgaaagatt
     3361 aaaagaaaag aaattctatt atctagtatt aaaataatac caataataag caacactttt
     3421 ttgttatttt ttattataaa gattaatttg ctactttaaa ttgaaaaaaa ctttacaggt
     3481 ttatacaaat tcgtacatca catgtataca atgctacata cattacttga taaatattac
     3541 gacgtgaaac gtattaattt aaacgaatct tagttatgta aataaaaaaa ggttagattt
     3601 tagtaaaaaa aaaagaccaa attttatatt aaatataaat cttgcattga atgcatttat
     3661 gtcaaattgc atgcaccttg ccgcccacca tgcatagacg cgcgccaata cgtattaata
     3721 gacgattaca tgacaaagtt tagagattaa ataaaaatca agtataaatt tagaatagac
     3781 aacatgtaaa aagtgagatg aatcttttta cgatacgtac agtgcgcatt aaaaatccaa
     3841 agtatatttt tattttcctg cgtgcgtata catatcttta aatacttgga taaagagata
     3901 ataagaattg tacggcaagc aaagtagtag tttaatagag cagtttttat attttatata
     3961 tagcttactt acaatataga tttttttttt tcatagattt cgggcatatt acacaagtaa
     4021 caaaagatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg
     4081 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     4141 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc
     4201 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     4261 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     4321 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     4381 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     4441 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     4501 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag
     4561 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc
     4621 tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc
     4681 accggcggca tggacgagct gtacaagtaa atcaatattg ttcttggagt tgttcttaac
     4741 agcttatttc ctgaacacag atacgtattg caacttaaat agaaatatga attacacaag
     4801 ttatcacttc ttgtagacaa tttcttatta cattattcag atctcagttc tattctcatt
     4861 tgaaagctaa aaaaaaagat gataaattgt ctaatcaatg ctaataaact ctaaatttag
     4921 caagaaaatt ctattataat tgttagtcaa tgataactaa tattatggat agaaggtggg
     4981 ctagtttgaa tcaaaatagt tactaagtaa tgtgtagttc gatacgattt tttccttctt
     5041 tttaaacgaa agtgtttaaa ttatgtgcgt gttgttttcc tgagcacagt attagaaatt
     5101 aattaagttg atttgtcagg tgatgtttat taccaaagtt caagtccgaa aatagagctg
     5161 ccatttaaca ataaaaaaga gcggtccaaa tttaggcaaa acaaattttt taaaaaaagc
     5221 tccaagtccg ggaattgaac ccgggtcatc cgcgtgacag gcggatatac taggtgggga
     5281 aactaaatat actgaaattc ggtagattct atatctcacg ggacagcttt tcacacacac
     5341 ttagttctat attgcgtcat aacttttgtt tttttttgtt gcactttttt tctcttatat
     5401 tcaagtaagt ggtttagatt ctctaagggc gggaatatga attagtggca attcaaagga
     5461 tttaaacgat caggcgcctg cacaccaaac tctaattcgt acacagcatg cccgaggtta
     5521 tagatataga tactgcgaaa ttattttcat tgtttcagtt aagaaataat aaaatatttt
     5581 attatagtta ttttccaaat ttatttgaga tttttgtatt gaagtttagc cgtcgacatg
     5641 gtcttcacac tcgaagattt cgttggggac tggcgacaga cagccggcta caacctggac
     5701 caagtccttg aacagggagg tgtgtccagt ttgtttcaga atctcggggt gtccgtaact
     5761 ccgatccaaa ggattgtcct gagcggtgaa aatgggctga agatcgacat ccatgtcatc
     5821 atcccgtatg aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat ttttaaggtg
     5881 gtgtaccctg tggatgatca tcactttaag gtgatcctgc actatggcac actggtaatc
     5941 gacggggtta cgccgaacat gatcgactat ttcggacggc cgtatgaagg catcgccgtg
     6001 ttcgacggca aaaagatcac tgtaacaggg accctgtgga acggcaacaa aattatcgac
     6061 gagcgcctga tcaaccccga cggctccctg ctgttccgag taaccatcaa cggagtgacc
     6121 ggctggcggc tgtgcgaacg cattctggcg gctagcggaa gcggagctac taacttcagc
     6181 ctgctgaagc aggctggaga cgtggaggag aaccctggac ctatgattga acaagatggt
     6241 ttacacgctg gttctcccgc cgcttgggtc gaaagacttt tcggttatga ctgggctcaa
     6301 caaaccatcg gttgctctga tgccgccgtc ttccgtcttt ctgctcaagg tcgtcctgtt
     6361 cttttcgtca agaccgacct ttctggtgcc cttaatgaac ttcaagatga agctgcccgt
     6421 ctttcttggc ttgccaccac cggtgttcct tgcgctgctg tccttgacgt tgtcactgaa
     6481 gccggtagag actggcttct tttaggtgaa gtccccggtc aagatcttct ttcttctcac
     6541 cttgctcctg ccgaaaaagt ttctatcatg gctgatgcta tgcgtcgtct tcataccctt
     6601 gatcccgcta cctgcccttt cgaccaccaa gccaaacatc gtatcgaacg tgctcgtact
     6661 cgtatggaag ccggtcttgt cgatcaagat gatcttgacg aagaacatca aggtcttgcc
     6721 cctgccgaac ttttcgccag acttaaggcc cgtatgcccg acggtgaaga tcttgtcgtc
     6781 acccatggtg atgcctgctt acccaatatc atggttgaaa atggtcgttt ttctggtttc
     6841 atcgactgtg gtcgtcttgg tgtcgccgac cgttatcaag atattgcctt agctacccgt
     6901 gatattgctg aagaacttgg tggtgaatgg gctgaccgtt tccttgtcct ttacggtatc
     6961 gccgctcccg attctcaacg tatcgccttc tatcgtcttc ttgacgaatt cttctgatta
     7021 attaagcgca tgcaatctat aactaattcc aaaattagtg aataaaaatg atagaaaaga
     7081 ctataagctc gaaattgtgt ttattacgta ctataatatg ccaatcttct tatatattga
     7141 aaagttatta tgaatttaat aaataaactt taccgaatat ataagagaga aaaaaaggag
     7201 gacttaagaa taagtgacaa agaagagcca ttaataatat caaaaacaag aatggcacta
     7261 ttagaactag aaattaaaac tttagcttac attctttcta gagaattaaa aacgagttct
     7321 caaatttcta atctatccga aatcaaatgg gcagaaatta ccactgatga cttggataga
     7381 cttgaatcca tcttgtttga aaatgtctac gaaatagaaa ttaacccgat agttaagtca
     7441 ttaatctcta ttttgagagg actattagat acacgtaaca agttttttaa tggcaaagcg
     7501 atgccgttct atgtaaaatt aaagaatgag aatgttcaaa gtacatttat tttagaagaa
     7561 agcgttaatt ttccatttag aaacgataga ttaatcaaac aatgtgaaaa tataaactct
     7621 agtaatgcta ctccaaaatc atatttctct tcaagaaccc caagaaacct tccagacttt
     7681 aatcaaaagg tagaattgcc aattaaaagt aggtcgaacc atataaattt aatttcttca
     7741 actccaagaa gtacttcgtg tcaaacacct aggacaaaga atcacatgaa acatacgaat
     7801 ttagattatt ctacaccaaa atccgaaata aataaaaaaa attattatat ttttagtgat
     7861 tcaaataccc caagaagaag ctcaaaaact ggaatttgtg gcaattcgaa aatatctaac
     7921 aaccagcaag gaagtataat cccgggtacc gagctcgaat tcactggccg tcgttttaca
     7981 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     8041 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     8101 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     8161 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     8221 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     8281 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     8341 atcaccgaaa cgcgcga
//
