LOCUS       sequence-264608-        8662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP protein and a fusion protein of Nluc-neo inserting 
            into Cryptosporidium parvum TK locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8662)
  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 8662)
  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: TK-GFP-Nluc-P2A-neo-TK
FEATURES             Location/Qualifiers
     source          1..8662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..19
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(57..79)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     179..198
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     392..414
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     406..423
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     407..423
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(2455..2457,2458..2460,2461..3174)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2500..2521)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3108..3129
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4101..4613
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             4629..4685
                     /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(6469..6485)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6469..6485)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6482..6504)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6493..6509
                     /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)."
     promoter        complement(join(6517..6523,6524..6541,6542..6547))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6562..6583
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6700..6717)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6871..7459)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6951..6970)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7630..8421,8422..8490))
                     /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     8253..8272
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8491..8595)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
       61 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
      121 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
      181 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
      241 gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa
      301 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
      361 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
      421 agtgaattcg agctgagctc catttgtcct aacttcatct atggtcttat ttttatcaac
      481 ctgcttaata aaagagttca aattcctatc tacaaaaatt acctggtctt catcatctat
      541 gtcatctttc agtaaatggc tttcaacaat taatgcagta ttgtctttat catcaatata
      601 ttctttatca atatcaatcc caatcattcc atttttacgt tttctacctt taaatccgcc
      661 ttcattaatt tcacaattat tgaaaaaatc ttcgtccaaa ctactatcat tacattcaag
      721 cttctcagtt aaaatattgt caactttatt ataaaatgta acattttgac aaactattgg
      781 cttttcaaag ttatactccc agtcaggcgg aatcacctca tcatctggta caacaaattc
      841 atcagagtct attgaaagtt tatcagaatc atctttcaat ttactttttt tcctccttgg
      901 tcttgttttc tttgacttag ctctacgctt tttcaattta tctggtgcat cttgattttg
      961 ttcatcgcca atccttttca tatagctaac tggattcaaa aaattggaat tttcctggta
     1021 ataaacttta tctaattcat ctgattcttc cctcaaatca acaaataata tttcctcttc
     1081 cttattatac ccacctaata ataattcaag atcctcctca gtaggcccat tttcaagact
     1141 acttatcata atttcttatc aaactcaagt taaaaagcat tatcacacca aaaacatata
     1201 ttctactcga aatcccactc ttatataata atatcaattt acatttcaag tatctgaaaa
     1261 tgaacccttt tggcgccttt taatatctat atataggtaa ccacttttct caagtaataa
     1321 atttgaggct aaattgagat taaataacta ctcataaagt aaaaataaat taaatttatt
     1381 tttattaatc actcaaaaca aaattttaaa ccatctaaca caaagaattc aacaatttac
     1441 ctaataaata ctttaaagca ataatatcac tcatacctca gaatgagttg gttataaaca
     1501 gtaataatag aaagcgttag ggctataaaa atgaccaaat aagtcattta gcatcataag
     1561 tatatacatt agatacagaa tgagttcaaa ataaataaaa aattagactt acttacaaat
     1621 tctaagctat agtataaaag aactaagaag atttgatgaa tttgctatac cattttaaat
     1681 atttgtttat agttttttga tattaatata aggactatta agtgaaaagg aaactacaca
     1741 tatttccgcc aattcattgt attgcatcgc acacataaat gaaagattaa aagaaaagaa
     1801 attctattat ctagtattaa aataatacca ataataagca acactttttt gttatttttt
     1861 attataaaga ttaatttgct actttaaatt gaaaaaaact ttacaggttt atacaaattc
     1921 gtacatcaca tgtatacaat gctacataca ttacttgata aatattacga cgtgaaacgt
     1981 attaatttaa acgaatctta gttatgtaaa taaaaaaagg ttagatttta gtaaaaaaaa
     2041 aagaccaaat tttatattaa atataaatct tgcattgaat gcatttatgt caaattgcat
     2101 gcaccttgcc gcccaccatg catagacgcg cgccaatacg tattaataga cgattacatg
     2161 acaaagttta gagattaaat aaaaatcaag tataaattta gaatagacaa catgtaaaaa
     2221 gtgagatgaa tctttttacg atacgtacag tgcgcattaa aaatccaaag tatattttta
     2281 ttttcctgcg tgcgtataca tatctttaaa tacttggata aagagataat aagaattgta
     2341 cggcaagcaa agtagtagtt taatagagca gtttttatat tttatatata gcttacttac
     2401 aatatagatt tttttttttc atagatttcg ggcatattac acaagtaaca aaagatggtg
     2461 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     2521 gtaaacggcc acaagttcag cgtgcgcggc gagggcgagg gcgatgccac caacggcaag
     2581 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     2641 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     2701 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat ctccttcaag
     2761 gacgacggca cctacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     2821 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     2881 gagtacaact tcaacagcca caacgtctat atcacggccg acaagcagaa gaacggcatc
     2941 aaggcgaact tcaagatccg ccacaacgtc gaggacggca gcgtgcagct cgccgaccac
     3001 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     3061 agcacccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     3121 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtagatcaat
     3181 attgttcttg gagttgttct taacagctta tttcctgaac acagatacgt attgcaactt
     3241 aaatagaaat atgaattaca caagttatca cttcttgtag acaatttctt attacattat
     3301 tcagatctca gttctattct catttgaaag ctaaaaaaaa agatgataaa ttgtctaatc
     3361 aatgctaata aactctaaat ttagcaagaa aattctatta taattgttag tcaatgataa
     3421 ctaatattat ggatagaagg tgggctagtt tgaatcaaaa tagttactaa gtaatgtgta
     3481 gttcgatacg attttttcct tctttttaaa cgaaagtgtt taaattatgt gcgtgttgtt
     3541 ttcctgagca cagtattaga aattaattaa gttgatttgt caggtgatgt ttattaccaa
     3601 agttcaagtc cgaaaataga gctgccattt aacaataaaa aagagcggtc caaatttagg
     3661 caaaacaaat tttttaaaaa aagctccaag tccgggaatt gaacccgggt catccgcgtg
     3721 acaggcggat ataccaggtg gggaaactaa atatactgaa attcggtaga ttctatatct
     3781 cacgggacag cttttcacac acacttagtt ctatattgcg tcataacttt tgtttttttt
     3841 gttgcacttt ttttctctta tattcaagta agtggtttag attctctaag ggcgggaata
     3901 tgaattagtg gcaattcaaa ggatttaaac gatcaggcgc ctgcacacca aactctaatt
     3961 cgtacacagc atgcccgagg ttatagatat agatactgcg aaattatttt cattgtttca
     4021 gttaagaaat aataaaatat tttattatag ttattttcca aatttatttg agatttttgt
     4081 attgaagttt agccgtcgac atggtcttca cactcgaaga tttcgttggg gactggcgac
     4141 agacagccgg ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc
     4201 agaatctcgg ggtgtccgta actccgatcc aaaggattgt cctgagcggt gaaaatgggc
     4261 tgaagatcga catccatgtc atcatcccgt atgaaggtct gagcggcgac caaatgggcc
     4321 agatcgaaaa aatttttaag gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc
     4381 tgcactatgg cacactggta atcgacgggg ttacgccgaa catgatcgac tatttcggac
     4441 ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt
     4501 ggaacggcaa caaaattatc gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc
     4561 gagtaaccat caacggagtg accggctggc ggctgtgcga acgcattctg gcggctagcg
     4621 gaagcggagc tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg
     4681 gacctatgat tgaacaagat ggtttacacg ctggttctcc cgccgcttgg gtcgaaagac
     4741 ttttcggtta tgactgggct caacaaacca tcggttgctc tgatgccgcc gtcttccgtc
     4801 tttctgctca aggtcgtcct gttcttttcg tcaagaccga cctttctggt gcccttaatg
     4861 aacttcaaga tgaagctgcc cgtctttctt ggcttgccac caccggtgtt ccttgcgctg
     4921 ctgtccttga cgttgtcact gaagccggta gagactggct tcttttaggt gaagtccccg
     4981 gtcaagatct tctttcttct caccttgctc ctgccgaaaa agtttctatc atggctgatg
     5041 ctatgcgtcg tcttcatacc cttgatcccg ctacctgccc tttcgaccac caagccaaac
     5101 atcgtatcga acgtgctcgt actcgtatgg aagccggtct tgtcgatcaa gatgatcttg
     5161 acgaagaaca tcaaggtctt gcccctgccg aacttttcgc cagacttaag gcccgtatgc
     5221 ccgacggtga agatcttgtc gtcacccatg gtgatgcctg cttacccaat atcatggttg
     5281 aaaatggtcg tttttctggt ttcatcgact gtggtcgtct tggtgtcgcc gaccgttatc
     5341 aagatattgc cttagctacc cgtgatattg ctgaagaact tggtggtgaa tgggctgacc
     5401 gtttccttgt cctttacggt atcgccgctc ccgattctca acgtatcgcc ttctatcgtc
     5461 ttcttgacga attcttctga ttaattaagg caaaatttgg cgcagctatg gcgcctcttg
     5521 aaagtgccta aaaaggagtc aaagttagac agaccaagaa aaggtaacaa ggcaagagaa
     5581 aacatacatt aatatatata catatatata tacttaatag agatagtatt actcttctat
     5641 tctcttgtat agattggtat tagtagtctt ttatggaaaa ggagaagaga tgaataaagg
     5701 ttacaaagga ggatgctttt ctctaaaagt accaccagtg ttttgcaata atgggaattt
     5761 attattaatc ccaaaaggaa gagataacga aatttatgta tatgatactt tgggaaatgg
     5821 agagtctaaa tctcaatttt ctgcttcaaa agaaattatc acaggtcttg gtgtgataaa
     5881 gtctgatcaa aatgaggaat ttattgttac tacaactgtt agtggtttaa tacgaatttt
     5941 tcatttaaat gatatcttaa atcataaaga aaagaagtct gtaatagaga ttgaaattgg
     6001 tcgatgtatt attgatttga aagttgcaaa aaacaatata tattttatca taagtgaagc
     6061 aaaagatcaa gatactttga ttgatactaa tgtttcctta tataaaattc ccatttctga
     6121 tataatttca aaatttaata cttctgttaa tcaaaaatct aatcattcca aatttattca
     6181 aaaatctctc aaaaagatta ttaacttctc atatggagct ttaaatttcc aagtatcttc
     6241 ggatgagtct ttattctgtt ttatatggaa aaatatctta attatttgga atactcaata
     6301 ccctgataaa attattcgtt ttagacattc cgaatatatt ctatcgctta cactttctga
     6361 agatcaacaa tttgttgcta ctggtgatgc ttatggtagg ggtaccagct cggtacccgg
     6421 ggatcctcta gagtcgacct gcaggcatgc aagcttggcg taatcatggt catagctgtt
     6481 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     6541 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     6601 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6661 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6721 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6781 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6841 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6901 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6961 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     7021 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     7081 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     7141 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     7201 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     7261 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     7321 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     7381 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     7441 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     7501 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     7561 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     7621 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7681 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7741 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7801 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7861 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7921 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7981 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     8041 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     8101 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     8161 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     8221 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     8281 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     8341 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     8401 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     8461 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     8521 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     8581 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     8641 tatcatgaca ttaacctata aa
//
