LOCUS       sequence-275776-        7551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP-tagged gene expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7551)
  AUTHORS   Yadav L, Pietila E, Ohman T, Liu X, Mahato AK, Sidorova Y, Lehti K, 
            Saarma M, Varjosalo M
  TITLE     PTPRA Phosphatase Regulates GDNF-Dependent RET Signaling and 
            Inhibits the RET Mutant MEN2A Oncogenic Potential.
  JOURNAL   iScience. 2020 Feb 21;23(2):100871. doi: 10.1016/j.isci.2020.100871.
            Epub 2020 Jan 31.
  PUBMED    32062451
REFERENCE   2  (bases 1 to 7551)
  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: "iScience.";
            date: "2020-02-21"; volume: "23(2):100871. doi"; pages: " 
            10.1016/j.isci.2020.100871. Epub 2020 Jan 31"
COMMENT     Sequence Label: PTPRA_deltaD1-GFP fusion
FEATURES             Location/Qualifiers
     source          1..7551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    802..826
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2432..2456)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2464..2473
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2470..2472,2473..2475,2476..3186)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2470..2472,2473..2475,2476..3186)
                     /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(2515..2536)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2776..2795)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3123..3144
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3269..3286)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3275..3499
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3632..3651)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3783..3830
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3838..4860
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3838..4860
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4990..5111
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5027..5046)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5081..5100
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5160..5176)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5160..5176)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5173..5195)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5184..5200
                     /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(5208..5214,5215..5232,5233..5238))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5253..5274
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5391..5408)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5562..6150)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5642..5661)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6321..7112,7113..7181))
                     /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     6944..6963
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7182..7286)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7361..7380)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagcttg gtaccgagct cggatccact agtccagtgt
      781 ggtggaattc tgcagatatc gacaagtttg tacaaaaaag caggctccac catggattcc
      841 tggttcattc ttgttctgct cggcagtggt ctgatatgtg tcagtgccaa caatgctacc
      901 acagttgcac cttctgtagg aattacaaga ttaattaact catcaacggc agaaccagtt
      961 aaagaagagg ccaaaacttc aaatccaact tcttcactaa cttctctttc tgtggcacca
     1021 acattcagcc caaatataac tctgggaccc acctatttaa ccactgtcaa ttcttcagac
     1081 tctgacaatg ggaccacaag aacagcaagc accaattcta taggcattac aatttcacca
     1141 aatggaacgt ggcttccaga taaccagttc acggatgcca gaacagaacc ctgggagggg
     1201 aattccagca ccgcagcaac cactccagaa actttccctc cttcagatga gacaccaatt
     1261 attgcggtga tggtggccct gtcctctctg ctagtgatcg tgtttattat catagttttg
     1321 tacatgttaa ggtttaagaa atacaagcaa gctgggagcc attccaattc tttccgctta
     1381 tccaacggcc gcactgagga tgtggagccc cagagtgtgc cacttctggc cagatcccca
     1441 agcaccaaca ggaaataccc acccctgccc gtggacaagc tggaagagga aattaaccgg
     1501 agaatggcag acgacaataa gctctatctc tatggagata cagaactgga agtgacctct
     1561 ctagaaaccc acctgcagaa aatttacaac aaaatcccag ggaccagcaa caatggatta
     1621 gaggaggagt ttaagaagtt aacatcaatc aaaatccaga atgacaagat gcggactgga
     1681 aaccttccag ccaacatgaa gaagaaccgt gttttacaga tcattccata tgaattcaac
     1741 agagtgatca ttccagttaa gcggggcgaa gagaatacag actatgtgaa cgcatccttt
     1801 attgatggct accggcagaa ggactcctat atcgccagcc agggccctct tctccacaca
     1861 attgaggact tctggcgaat gatctgggag tggaaatcct gctctatcgt gatgctaaca
     1921 gaactggagg agagaggcca ggagaagtgt gcccagtact ggccatctga tggactggtg
     1981 tcctatggag atattacagt ggaactgaag aaggaggagg aatgtgagag ctacaccgtc
     2041 cgagacctcc tggtcaccaa caccagggag aataagagcc ggcagatccg gcagttccac
     2101 ttccatggct ggcctgaagt gggcatcccc agtgacggaa agggcatgat cagcatcatc
     2161 gccgccgtgc agaagcagca gcagcagtca gggaaccacc ccatcaccgt gcactgcagc
     2221 gccggggcag gaaggacggg gaccttctgt gccctgagca ccgtcctgga gcgtgtgaaa
     2281 gcagagggga ttttggatgt cttccagact gtcaagagcc tgcggctaca gaggccacac
     2341 atggtccaga cactggaaca gtatgagttc tgctacaagg tggtgcagga gtatattgat
     2401 gcattctcag attatgccaa cttcaagttg gacccagctt tcttgtacaa agtggtgagc
     2461 tccgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     2521 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     2581 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     2641 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     2701 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     2761 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2821 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2881 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     2941 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     3001 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     3061 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     3121 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     3181 tacaagcacc ggtccacgtg acgtaagcta gatccagcac agtggcggcc gctcgagtct
     3241 agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     3301 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     3361 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     3421 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     3481 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     3541 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3601 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3661 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3721 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc
     3781 tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc cttggccaaa
     3841 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     3901 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     3961 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     4021 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     4081 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     4141 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     4201 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     4261 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     4321 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     4381 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     4441 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     4501 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     4561 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     4621 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     4681 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     4741 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     4801 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag
     4861 cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4921 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4981 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5041 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5101 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5161 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5221 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5281 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5341 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5401 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5461 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5521 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5581 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5641 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5701 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5761 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5821 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5881 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5941 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6001 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6061 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6121 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6181 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6241 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6301 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6361 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6421 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6481 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6541 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6601 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6661 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6721 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6781 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6841 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6901 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6961 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7021 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7081 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7141 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7201 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7261 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     7321 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     7381 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     7441 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
     7501 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt t
//
