LOCUS       pcDNA5-FRT_TO_3x        6523 bp DNA     circular SYN 22-MAY-2024
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6523)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 22, 2024 from SnapGene Viewer 6.2.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          1..1075
                     /label=source
                     /note="/dnas_title=Restriction 1 Restriction Clone"
                     /note="color: #c6c9d1"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #75c6a9"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate earlypromoter"
                     /note="color: #c7b0e3"
     protein_bind    820..838
                     /label=tetO protein_bind
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="/dnas_title=tetO"
                     /note="color: #75c6a9"
     protein_bind    841..859
                     /label=tetO protein_bind
                     /bound_moiety="tetracycline repressor TetR"
                     /note="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
                     /note="/dnas_title=tetO"
                     /note="color: #b1ff67"
     misc_feature    979..1075
                     /label=3xFLAG insert from annealed oligos
                     /note="color: #d59687"
     CDS             993..2414
                     /codon_start=1
                     /label=3xFLAG-NESmut-NFATC2IP delM
                     /note="color: #b7e6d7"
                     /translation="DYKDHDGDYKDHDIDYKDDDDKAASLNGAPNLVDLQKKLEEAKAD
                     EQQGAGAGAPAEPVGKRGRWSGGSGAGRGGRGGWGGRGRRPRAQRSPSRGTLDVVSVDL
                     VTDSDEEILEVATARGAADEVEVEPPEPPGPVASRDNSNSDSEGEDRRPAGPPREPVRR
                     RRRLVLDPGEAPLVPVYSGKVKSSLRLIPDDLSLLKLYPPGDEEEAELADSSGLYHEGS
                     PSPGSPWKTKLRTKDKEEKKKTEFLDLDNSPLSPPSPRTKSRTHTRALKKLSEVNKRLQ
                     DLRSCLSPKPPQGQEQQGQEDEVVLVEGPTLPETPRLFPLKIRCRADLVRLPLRMSEPL
                     QSVVDHMATHLGVSPSRILLLFGETELSPTATPRTLKLGVADIIDCVVLTSSPEATETS
                     QQLQLRVQGKEKHQTLEVSLSRDSPLKTLMSHYEEAMGLSGRKLSFFFDGTKLSGRELP
                     ADLGMESGDLIEVWG"
     CDS             993..1058
                     /codon_start=1
                     /label=3xFLAG
                     /note="color: #f58a5e"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1083..1136
                     /codon_start=1
                     /label=mutant NES (LEL to AKA)
                     /note="color: #ff9ccd"
                     /translation="NLVDLQKKLEEAKADEQQ"
     misc_feature    1116..1124
                     /label=LEL to AKA mutation
                     /note="color: #c7b0e3"
     CDS             1158..2414
                     /codon_start=1
                     /label=NFATC2IP delMet ORF
                     /note="color: #ffef86"
                     /translation="AEPVGKRGRWSGGSGAGRGGRGGWGGRGRRPRAQRSPSRGTLDVV
                     SVDLVTDSDEEILEVATARGAADEVEVEPPEPPGPVASRDNSNSDSEGEDRRPAGPPRE
                     PVRRRRRLVLDPGEAPLVPVYSGKVKSSLRLIPDDLSLLKLYPPGDEEEAELADSSGLY
                     HEGSPSPGSPWKTKLRTKDKEEKKKTEFLDLDNSPLSPPSPRTKSRTHTRALKKLSEVN
                     KRLQDLRSCLSPKPPQGQEQQGQEDEVVLVEGPTLPETPRLFPLKIRCRADLVRLPLRM
                     SEPLQSVVDHMATHLGVSPSRILLLFGETELSPTATPRTLKLGVADIIDCVVLTSSPEA
                     TETSQQLQLRVQGKEKHQTLEVSLSRDSPLKTLMSHYEEAMGLSGRKLSFFFDGTKLSG
                     RELPADLGMESGDLIEVWG"
     polyA_signal    2481..2705
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #f58a5e"
     protein_bind    2989..3036
                     /label=FRT
                     /bound_moiety="FLP recombinase from the 
                     Saccharomycescerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp 
                     coresequence TCTAGAAA (Turan and Bode, 2011)."
                     /note="color: #f8d3a9"
     CDS             3044..4066
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /note="color: #ccffcc"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGDFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4196..4317
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #d59687"
     primer_bind     complement(4366..4382)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple 
                     similarvariants"
                     /note="color: #84b0dc; direction: LEFT"
     protein_bind    4390..4406
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator 
                     toinhibit transcription in E. coli. This inhibition can 
                     berelieved by adding lactose 
                     orisopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /note="color: #84b0dc"
     promoter        complement(4414..4444)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="color: #75c6a9; direction: LEFT"
     protein_bind    4459..4480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in thepresence 
                     of cAMP."
                     /note="color: #b1ff67"
     rep_origin      complement(4768..5356)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin 
                     ofreplication"
                     /note="/dnas_title=ori"
                     /note="color: #b1ff67"
     CDS             complement(5527..6387)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 5527 .. 6318 / #ccffcc
                        2: 6319 .. 6387 / #ccffcc / signal sequence
                     Cleavage site after base 6318"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6388..6492)
                     /label=bla promoter
                     /note="AmpR promoter"
                     /note="/dnas_title=AmpR promoter"
                     /note="color: #b7e6d7; direction: LEFT"
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtacca tggactacaa ggaccacgac ggtgactaca
     1021 aggaccacga catcgactac aaagacgatg acgataaagc cgccagttta aacggcgcgc
     1081 caaatctcgt ggacctccaa aagaagctgg aggaggccaa ggctgacgag cagcagggcg
     1141 ctggagccgg cgcgccagcg gagcctgtgg ggaagcgggg ccgctggtcc ggaggtagcg
     1201 gtgccggccg agggggtcgg ggcggctggg gcggtcgggg ccggcgtcct cgggcccagc
     1261 ggtctccatc ccggggcacg ctggacgtag tgtctgtgga cttggtcacc gacagcgatg
     1321 aggaaattct ggaggtcgcc accgctcgcg gtgccgcgga cgaggttgag gtggagcccc
     1381 cggagccccc ggggccggtc gcgtcccggg ataacagcaa cagtgacagc gaaggggagg
     1441 acaggcggcc cgcaggaccc ccgcgggagc cggtcaggcg gcggcggcgg ctggtgctgg
     1501 atccggggga ggcgccgctg gttccggtgt actcggggaa ggttaaaagc agccttcgcc
     1561 ttatcccaga tgatctatcc ctcctgaaac tctaccctcc aggggatgag gaagaggcag
     1621 agctggcaga ttcgagtggt ctctaccatg agggctcccc atcaccaggc tctccctgga
     1681 agacaaagct gaggactaag gataaagaag agaagaaaaa gacagagttt ctggatctgg
     1741 acaactctcc tctgtcccca ccttcaccaa ggaccaaaag cagaacgcat actcgggcac
     1801 tcaagaagtt aagtgaggtg aacaagcgcc tccaggatct ccgttcctgt ctgagcccca
     1861 agccacctca gggtcaagag caacagggcc aagaggatga agtggtcttg gtggaagggc
     1921 ccaccctccc agagaccccc cgactcttcc cactcaaaat ccgttgccgg gctgacctgg
     1981 tcagattgcc cctcaggatg tcggagcccc tgcagagtgt ggtggaccac atggccaccc
     2041 accttggggt gtccccaagc aggatccttt tgctttttgg agagacagag ctatcaccta
     2101 ctgccactcc caggacccta aagctcggag tggctgacat cattgactgt gtggtactaa
     2161 caagttctcc agaggccaca gagacgtccc aacagctcca gctccgggtg cagggaaagg
     2221 agaaacacca gacactggaa gtctcactgt ctcgagattc ccctctaaag accctcatgt
     2281 cccactatga ggaggccatg ggactgtcgg gacggaagct ctccttcttc tttgatggga
     2341 caaagctttc aggcagggag ctgccagctg acctgggcat ggaatctggg gacctcattg
     2401 aggtctgggg ctgagatatc catcacactg gcggccgctc gagtctagag ggcccgttta
     2461 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     2521 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     2581 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     2641 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     2701 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     2761 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2821 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2881 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2941 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt
     3001 ccgaagttcc tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac
     3061 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
     3121 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     3181 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
     3241 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggatttca gcgagagcct
     3301 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga
     3361 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct
     3421 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
     3481 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
     3541 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
     3601 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
     3661 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
     3721 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
     3781 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct
     3841 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
     3901 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
     3961 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
     4021 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga
     4081 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4141 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     4201 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4261 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4321 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     4381 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4441 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4501 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4561 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4621 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4681 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4741 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4801 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4861 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4921 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4981 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5041 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5101 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5161 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5221 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5281 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5341 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5401 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5461 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5521 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5581 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5641 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5701 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5761 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5821 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5881 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5941 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6001 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6061 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6121 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6181 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6241 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6301 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6361 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6421 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6481 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
