LOCUS       sequence-266476-        8356 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 8356)
  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 8356)
  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..8356
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(668..686)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     936..955
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2160..2672
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             2688..2744
                     /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(4485..4502)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4485..4501)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4494..4516)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4710..4729)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4829..4851
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4889..4907)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4975..5079
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5080..5148,5149..5940)
                     /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(5298..5317)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6111..6699
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6600..6619
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6853..6870
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6987..7008
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7023..7028,7029..7046,7047..7053)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7061..7077
                     /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     7066..7088
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7085..7101
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7085..7101
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ggtttataca aattcgtaca tcacatgtat acaatgctac atacattact tgataaatat
       61 tacgacgtga aacgtattaa tttaaacgaa tcttagttat gtaaataaaa aaaggttaga
      121 ttttagtaaa aaaaaaagac caaattttat attaaatata aatcttgcat tgaatgcatt
      181 tatgtcaaat tgcatgcacc ttgccgccca ccatgcatag acgcgcgcca atacgtatta
      241 atagacgatt acatgacaaa gtttagagat taaataaaaa tcaagtataa atttagaata
      301 gacaacatgt aaaaagtgag atgaatcttt ttacgatacg tacagtgcgc attaaaaatc
      361 caaagtatat ttttattttc ctgcgtgcgt atacatatct ttaaatactt ggataaagag
      421 ataataagaa ttgtacggca agcaaagtag tagtttaata gagcagtttt tatattttat
      481 atatagctta cttacaatat agattttttt ttttcataga tttcgggcat attacacaag
      541 taacaaaaga tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
      601 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
      661 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
      721 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
      781 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
      841 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
      901 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
      961 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     1021 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     1081 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     1141 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     1201 tccaccggcg gcatggacga gctgtacaag taaatcaata ttgttcttgg agttgttctt
     1261 aacagcttat ttcctgaaca cagatacgta ttgcaactta aatagaaata tgaattacac
     1321 aagttatcac ttcttgtaga caatttctta ttacattatt cagatctcag ttctattctc
     1381 atttgaaagc taaaaaaaaa gatgataaat tgtctaatca atgctaataa actctaaatt
     1441 tagcaagaaa attctattat aattgttagt caatgataac taatattatg gatagaaggt
     1501 gggctagttt gaatcaaaat agttactaag taatgtgtag ttcgatacga ttttttcctt
     1561 ctttttaaac gaaagtgttt aaattatgtg cgtgttgttt tcctgagcac agtattagaa
     1621 attaattaag ttgatttgtc aggtgatgtt tattaccaaa gttcaagtcc gaaaatagag
     1681 ctgccattta acaataaaaa agagcggtcc aaatttaggc aaaacaaatt ttttaaaaaa
     1741 agctccaagt ccgggaattg aacccgggtc atccgcgtga caggcggata tactaggtgg
     1801 ggaaactaaa tatactgaaa ttcggtagat tctatatctc acgggacagc ttttcacaca
     1861 cacttagttc tatattgcgt cataactttt gttttttttg ttgcactttt tttctcttat
     1921 attcaagtaa gtggtttaga ttctctaagg gcgggaatat gaattagtgg caattcaaag
     1981 gatttaaacg atcaggcgcc tgcacaccaa actctaattc gtacacagca tgcccgaggt
     2041 tatagatata gatactgcga aattattttc attgtttcag ttaagaaata ataaaatatt
     2101 ttattatagt tattttccaa atttatttga gatttttgta ttgaagttta gccgtcgaca
     2161 tggtcttcac actcgaagat ttcgttgggg actggcgaca gacagccggc tacaacctgg
     2221 accaagtcct tgaacaggga ggtgtgtcca gtttgtttca gaatctcggg gtgtccgtaa
     2281 ctccgatcca aaggattgtc ctgagcggtg aaaatgggct gaagatcgac atccatgtca
     2341 tcatcccgta tgaaggtctg agcggcgacc aaatgggcca gatcgaaaaa atttttaagg
     2401 tggtgtaccc tgtggatgat catcacttta aggtgatcct gcactatggc acactggtaa
     2461 tcgacggggt tacgccgaac atgatcgact atttcggacg gccgtatgaa ggcatcgccg
     2521 tgttcgacgg caaaaagatc actgtaacag ggaccctgtg gaacggcaac aaaattatcg
     2581 acgagcgcct gatcaacccc gacggctccc tgctgttccg agtaaccatc aacggagtga
     2641 ccggctggcg gctgtgcgaa cgcattctgg cggctagcgg aagcggagct actaacttca
     2701 gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acctatgatt gaacaagatg
     2761 gtttacacgc tggttctccc gccgcttggg tcgaaagact tttcggttat gactgggctc
     2821 aacaaaccat cggttgctct gatgccgccg tcttccgtct ttctgctcaa ggtcgtcctg
     2881 ttcttttcgt caagaccgac ctttctggtg cccttaatga acttcaagat gaagctgccc
     2941 gtctttcttg gcttgccacc accggtgttc cttgcgctgc tgtccttgac gttgtcactg
     3001 aagccggtag agactggctt cttttaggtg aagtccccgg tcaagatctt ctttcttctc
     3061 accttgctcc tgccgaaaaa gtttctatca tggctgatgc tatgcgtcgt cttcataccc
     3121 ttgatcccgc tacctgccct ttcgaccacc aagccaaaca tcgtatcgaa cgtgctcgta
     3181 ctcgtatgga agccggtctt gtcgatcaag atgatcttga cgaagaacat caaggtcttg
     3241 cccctgccga acttttcgcc agacttaagg cccgtatgcc cgacggtgaa gatcttgtcg
     3301 tcacccatgg tgatgcctgc ttacccaata tcatggttga aaatggtcgt ttttctggtt
     3361 tcatcgactg tggtcgtctt ggtgtcgccg accgttatca agatattgcc ttagctaccc
     3421 gtgatattgc tgaagaactt ggtggtgaat gggctgaccg tttccttgtc ctttacggta
     3481 tcgccgctcc cgattctcaa cgtatcgcct tctatcgtct tcttgacgaa ttcttctgat
     3541 taattaagcg catgcaatct ataactaatt ccaaaattag tgaataaaaa tgatagaaaa
     3601 gactataagc tcgaaattgt gtttattacg tactataata tgccaatctt cttatatatt
     3661 gaaaagttat tatgaattta ataaataaac tttaccgaat atataagaga gaaaaaaagg
     3721 aggacttaag aataagtgac aaagaagagc cattaataat atcaaaaaca agaatggcac
     3781 tattagaact agaaattaaa actttagctt acattctttc tagagaatta aaaacgagtt
     3841 ctcaaatttc taatctatcc gaaatcaaat gggcagaaat taccactgat gacttggata
     3901 gacttgaatc catcttgttt gaaaatgtct acgaaataga aattaacccg atagttaagt
     3961 cattaatctc tattttgaga ggactattag atacacgtaa caagtttttt aatggcaaag
     4021 cgatgccgtt ctatgtaaaa ttaaagaatg agaatgttca aagtacattt attttagaag
     4081 aaagcgttaa ttttccattt agaaacgata gattaatcaa acaatgtgaa aatataaact
     4141 ctagtaatgc tactccaaaa tcatatttct cttcaagaac cccaagaaac cttccagact
     4201 ttaatcaaaa ggtagaattg ccaattaaaa gtaggtcgaa ccatataaat ttaatttctt
     4261 caactccaag aagtacttcg tgtcaaacac ctaggacaaa gaatcacatg aaacatacga
     4321 atttagatta ttctacacca aaatccgaaa taaataaaaa aaattattat atttttagtg
     4381 attcaaatac cccaagaaga agctcaaaaa ctggaatttg tggcaattcg aaaatatcta
     4441 acaaccagca aggaagtata atcccgggta ccgagctcga attcactggc cgtcgtttta
     4501 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     4561 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     4621 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4681 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4741 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     4801 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     4861 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     4921 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     4981 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5041 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5101 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5161 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     5221 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5281 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5341 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5401 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5461 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5521 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5581 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5641 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5701 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5761 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5821 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5881 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5941 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6001 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6061 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6121 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6181 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6241 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6301 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6361 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6421 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6481 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6541 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6601 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6661 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6721 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6781 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     6841 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     6901 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     6961 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     7021 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     7081 cacacaggaa acagctatga ccatgattac gccaagcttg catgcctgca gagttataag
     7141 caccaagcta aacaaatatt aaaaagcatc gggaatgatc aaggagtaga aaaatgttct
     7201 attgatagtg gaaattatgt cttccactac attgtaaatg atggtattat ttatatgact
     7261 ttatcgggaa agtcataccc gaagaagttg gctttttctt ttctagatga aataaaaagg
     7321 ttatttattg aagaactagg gagagaattt gcaaatagag atgaattcaa gagcctgatt
     7381 gatgcaatag ataagccgta ttatttcata aagtttgacc gaataattca acgatgcaaa
     7441 tccgaatata aggacccaaa ctcgaacaag tcgctacaaa aactaaatga ctcgctaatt
     7501 gaggtatgtt tactacttgt ttttaaccat aatgctcaaa aaaattaaca attcaggtaa
     7561 ctaatataat gagaagaaat gtggatgata ttttactgag aggtgaaaat cttaatgatg
     7621 ttgaaagaaa agccaatgat ttaaaatttg catccctgga attttctaga gctgcaaaaa
     7681 agctttcatt gcatgcatta attcaaaaat atgcaccaat attggcctta tcggctatag
     7741 ttttgatcat cattatatgg aagcttattt tataaattca aatttctaaa ttgcggaata
     7801 aaaaaaactt tgaacattag cgccataact ttttttttga aaaaattttt gcaagtatta
     7861 cgtttaaaag tgctttataa attttactta aaatcaatag taaactcaat tgagagcttt
     7921 caatcagctc agaatgagtt ggttataaac agtaataata gaaagcgtta gggctataaa
     7981 aatgaccaaa taagtcattt agcatcataa gtatatacat tagatacaga atgagttcaa
     8041 aataaataaa aaattagact tacttacaaa ttctaagcta tagtataaaa gaactaagaa
     8101 gatttgatga atttgctata ccattttaaa tatttgttta tagttttttg atattaatat
     8161 aaggactatt aagtgaaaag gaaactacac atatttccgc caattcattg tattgcatcg
     8221 cacacataaa tgaaagatta aaagaaaaga aattctatta tctagtatta aaataatacc
     8281 aataataagc aacacttttt tgttattttt tattataaag attaatttgc tactttaaat
     8341 tgaaaaaaac tttaca
//
