LOCUS       sequence-72325-e        8353 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing AP-Smo-YFP in mammalian cells, can be used for 
            establishing Flp-In stable cell lines .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8353)
  AUTHORS   Ye F, Breslow DK, Koslover EF, Spakowitz AJ, Nelson WJ, Nachury MV
  TITLE     Single molecule imaging reveals a major role for diffusion in the 
            exploration of ciliary space by signaling receptors.
  JOURNAL   Elife. 2013 Aug 6;2:e00654. doi: 10.7554/eLife.00654. Print 2013.
  PUBMED    23930224
REFERENCE   2  (bases 1 to 8353)
  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: "Elife."; 
            date: "2013-08-6"; volume: "2:e00654. doi"; pages: " 
            10.7554/eLife.00654. Print 2013"
COMMENT     Sequence Label: pEF5B-FRT-AP-Smo-YFP-DEST
FEATURES             Location/Qualifiers
     source          1..8353
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        353..1531
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          584..1522
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1479..1499
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1548..1567
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1548..1566
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1645..1669
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1843..1887
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     regulatory      4123..4132
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4129..4131,4132..4134,4135..4845)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDEL*"
     primer_bind     complement(4174..4195)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4435..4454)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4782..4803
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4845..4869)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4922..4963
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(5001..5018)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5007..5231
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5364..5383)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    5515..5562
                     /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             5570..5965
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    6028..6147
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6063..6082)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6117..6136
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6196..6212)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6196..6212)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6209..6231)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6220..6236
                     /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(6244..6250,6251..6268,6269..6274))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6289..6310
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6427..6444)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6598..7186)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6678..6697)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7357..8148,8149..8217))
                     /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     7980..7999
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8218..8322)
                     /gene="bla"
                     /label=AmpR promoter
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 taggcttttg caaaaagctt tgcaaagatg gataaagttt taaacagaga
      301 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgcctcgt gaggctccgg
      361 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
      421 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
      481 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
      541 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg
      601 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac
      661 ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc
      721 gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg
      781 ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa
      841 gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag
      901 tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg
      961 cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc
     1021 accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc
     1081 gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg
     1141 agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg
     1201 ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc
     1261 cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc
     1321 gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc
     1381 ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt
     1441 ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca
     1501 aagttttttt cttccatttc aggtgtcgtg aggaattagc ttggtactaa tacgactcac
     1561 tatagggaga cccaagctgg ctaggtaagc ttggtaccga gctcggatcc actagtccag
     1621 tgtggtggaa ttctgcagat atcaacaagt ttgtacaaaa aagttgatat ggccgctggc
     1681 cgccccgtgc gtgggcccga gctggcgccc cggaggctgc tgcagttgct gctgctggta
     1741 ctgctggggg gcccgggccg gggggcggcc ttgagcggga acgtgaccgg gcctgggcct
     1801 cacagcgcca gcgggagctc gaggaggaac gtgccggtga ccggcctgaa cgatatcttc
     1861 gaggcccaga agatcgagtg gcacgaggtg accagccctc cgccgccgct gctgagccac
     1921 tgcggccggg ccgcccactg cgagcctttg cgctacaacg tgtgcctggg ctcggcgctg
     1981 ccctacggag ccaccaccac gctgctggct ggggactcgg actcgcagga ggaagcgcac
     2041 ggcaagctcg tgctctggtc cggcctccgg aatgcccccc gctgctgggc agtgatccag
     2101 cccctgctgt gtgctgtcta catgcccaag tgtgagaatg accgagtgga gttgcccagc
     2161 cgtaccctct gccaggccac ccgaggcccc tgtgccattg tggagcggga gcgagggtgg
     2221 cctgactttc tgcgttgcac accggaccac ttccctgaag gctgcccaaa cgaggtacaa
     2281 aacatcaagt tcaacagctc aggccaatgt gaagcaccct tggtgcgaac agacaacccc
     2341 aagagctggt atgaggacgt ggagggctgt gggattcagt gtcagaaccc gctgttcacc
     2401 gaggccgagc accaggacat gcacagctac atcgcagcct tcggggcggt cactggtctc
     2461 tgcacgctct tcacactggc cacctttgtg gctgactggc ggaattccaa tcgctaccct
     2521 gcggttattc tcttctatgt caatgcgtgt ttcttcgtgg gcagcattgg ctggctggcc
     2581 cagttcatgg atggtgcccg ccgagagatt gtttgccgag cagatggcac catgagattt
     2641 ggggagccca cctccagtga gaccctgtcc tgtgtcatca tctttgtcat tgtgtactat
     2701 gccttgatgg ctggagtagt ctggttcgtg gtcctcacct atgcctggca cacctccttc
     2761 aaagccctgg gcaccaccta ccagcctctc tcgggcaaga catcctattt ccacctgctc
     2821 acgtggtcac tcccctttgt cctcacggtg gcaatcctgg ctgtggctca ggtagatgga
     2881 gactccgtga gtggcatctg ttttgtaggc tacaagaact atcggtaccg tgctggcttt
     2941 gtcctggccc caattggcct ggtgcttatt gtgggaggct acttcctcat cagaggggtc
     3001 atgactctgt tctccatcaa gagcaaccac cctgggctct taagtgagaa ggcagccagc
     3061 aagatcaacg agaccatgct gcgcctgggc atttttggct tcctggcctt tggctttgtg
     3121 ctcatcacct tcagctgcca cttctatgac ttcttcaacc aggctgagtg ggagcgtagc
     3181 ttccgggact atgtgctatg ccaagccaac gtgaccatcg ggctgcctac caagaagccc
     3241 attcctgact gtgagatcaa gaatcggccc agcctcctgg tggagaagat caatctattt
     3301 gccatgtttg gcactggcat tgccatgagc acctgggtct ggaccaaggc caccctgctc
     3361 atctggaggc gcacctggtg caggttgact gggcacagtg atgatgagcc caagagaatc
     3421 aagaagagca agatgatcgc caaggccttc tctaagcggc gtgagctgct gcagaacccg
     3481 ggccaggagc tctccttcag catgcacact gtctcccatg atggacctgt tgcgggtttg
     3541 gcttttgacc tcaatgaacc ctcagctgac gtctcctctg cctgggctca gcatgtcacc
     3601 aagatggtgg ctcggagagg agccatattg ccccaggatg tgtccgttac ccctgtggca
     3661 actccagtgc caccagaaga acaagccaac atgtggctgg ttgaggcaga gatctcccca
     3721 gagttagaga agcgtttggg ccggaagaaa aagcggagga agaggaagaa ggaggtgtgc
     3781 cccttggggc cagcccctga gcttcaccac tctgcccctg ttcctgccac cagtgcagtt
     3841 cctcggctgc ctcagctgcc tcggcagaag tgcctggtag ctgcaaacgc ctggggaaca
     3901 ggggagtcct gccgacaggg agcctggact ctagtctcca accccttctg cccagagcct
     3961 agtccccatc aagatccatt tctccctggt gcctcagccc cccgggtctg ggctcagggc
     4021 cgcctccagg ggctgggatc cattcattcc cgcactaacc taatggaggc tgagatcttg
     4081 gatgcagact cggacttcag aaaagagcta gcgctaccgg tcgccaccat ggtgagcaag
     4141 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     4201 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     4261 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     4321 ttcggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     4381 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     4441 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     4501 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     4561 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     4621 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     4681 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     4741 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     4801 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt agtaggacaa ctttcttgta
     4861 caaagtggtt gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgcggttcga
     4921 aggtaagcct atccctaacc ctctcctcgg tctcgattct acgcgtaccg gttagtaatg
     4981 agtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     5041 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     5101 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     5161 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     5221 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     5281 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     5341 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     5401 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     5461 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta cctagaagtt
     5521 cctattccga agttcctatt ctctagaaag tataggaact tccttggccg ccaagccttt
     5581 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat
     5641 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg
     5701 tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc
     5761 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg
     5821 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa
     5881 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc
     5941 ctctggttat gtgtgggagg gctaagcaca attcgagctc ggtaccttta agaccaatga
     6001 cttacaaggc agctgtagat cttagccaac ttgtttattg cagcttaatg gttacaaata
     6061 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     6121 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     6181 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     6241 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     6301 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6361 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     6421 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     6481 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     6541 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     6601 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     6661 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6721 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6781 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6841 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6901 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6961 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     7021 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     7081 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     7141 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     7201 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     7261 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     7321 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     7381 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     7441 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     7501 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     7561 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     7621 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     7681 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7741 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7801 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7861 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7921 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     7981 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     8041 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     8101 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     8161 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     8221 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     8281 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     8341 gccacctgac gtc
//
