LOCUS       sequence-72324-e        7240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressing AP-Sstr3-GFP 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 7240)
  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 7240)
  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-Sstr3-GFP-DEST
FEATURES             Location/Qualifiers
     source          1..7240
                     /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             1672..1716
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     regulatory      3007..3016
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3013..3015,3016..3018,3019..3732)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3013..3015,3016..3018,3019..3729)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3058..3079)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3319..3338)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3666..3687
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3732..3756)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3809..3850
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3888..3905)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3894..4118
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4251..4270)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4402..4449
                     /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             4457..4852
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4915..5034
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4950..4969)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5004..5023
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5083..5099)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5083..5099)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5096..5118)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5107..5123
                     /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(5131..5137,5138..5155,5156..5161))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5176..5197
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5314..5331)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5485..6073)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5565..5584)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6244..7035,7036..7104))
                     /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     6867..6886
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7105..7209)
                     /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 gggcctgaac
     1681 gacatcttcg aggctcagaa aatcgaatgg cacgaagcca ctgttaccta tccctcatcc
     1741 gagcctatga ccttggaccc tgggaacaca tcctcgacct ggcccctgga taccaccctg
     1801 gggaacacat ccgctggcgc tagcctgaca ggcctggctg tcagtggcat cttgatctct
     1861 ctggtgtacc tggtggtgtg cgtggtgggt ctgctgggca actcgctggt gatctacgtg
     1921 gtcctgcggc acacgtccag cccatcagtg accagtgtct atatcctcaa cctggctctg
     1981 gctgatgagc tcttcatgct agggctaccc ttcctggctg ctcagaacgc cctgtcctac
     2041 tggccctttg gatctctcat gtgccgtctg gtcatggccg tggatggcat caaccagttc
     2101 accagcatct tctgcctcac cgtcatgagt gtggaccgct atctggctgt ggtgcacccc
     2161 acacgctcag cccgctggcg cacggcacca gtggctcgca cggtcagtgc agctgtctgg
     2221 gtggcctcgg ctgtggtggt gctgcctgtg gttgtgttct caggagtgcc ccggggcatg
     2281 agcacgtgcc acatgcagtg gccagagcca gcggctgcct ggcgaacagc ctttatcatc
     2341 tacacggccg cactgggctt ctttgggccc ctgctggtca tctgcttgtg ctacttgctc
     2401 attgtggtaa aggtgcggtc gaccacccgg cgggtgcggg cgccctcgtg tcagtgggta
     2461 caggcacccg catgccagcg gcgacgccgc tctgagcgca gggtcacacg catggtggtg
     2521 gccgtggtgg cactcttcgt cctctgctgg atgcctttct atctgctcaa catcgtcaat
     2581 gtggtgtgcc cgctgccgga ggagcccgcc ttcttcggcc tctacttcct ggtggtggcg
     2641 ctgccctacg ccaacagctg cgcaaacccc atcctctacg gcttcctctc ctaccgcttc
     2701 aagcagggct ttcgcaggat cctgctaaga ccatcacgtc gcattcggag ccaggagcca
     2761 gggtcgggac ctccagagaa gactgaagag gaggaggatg aagaagaaga agagagaagg
     2821 gaagaggagg agcggaggat gcagagaggg caggagatga acgggaggct cagtcagatc
     2881 gcacaggctg gcactagtgg acaacagcca cggccctgca cagggactgc taaggagcag
     2941 cagcttctgc cccaggaggc cacagctggg gacaaggcca gcacactgag ccatctggga
     3001 tccatcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     3061 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     3121 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     3181 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     3241 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     3301 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     3361 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     3421 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     3481 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     3541 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     3601 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     3661 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     3721 ctgtacaagt aagacaactt tcttgtacaa agtggttgat atccagcaca gtggcggccg
     3781 ctcgagtcta gagggcccgc ggttcgaagg taagcctatc cctaaccctc tcctcggtct
     3841 cgattctacg cgtaccggtt agtaatgagt ttaaacccgc tgatcagcct cgactgtgcc
     3901 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3961 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4021 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4081 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     4141 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4201 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4261 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4321 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4381 gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat
     4441 aggaacttcc ttggccgcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc
     4501 aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct
     4561 ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg
     4621 tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat
     4681 cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt
     4741 gcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac
     4801 ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct aagcacaatt
     4861 cgagctcggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccaacttg
     4921 tttattgcag cttaatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4981 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     5041 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5101 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5161 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5221 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5281 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5341 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5401 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5461 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5521 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5581 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5641 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5701 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5761 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5821 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5881 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     5941 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6001 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6061 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6121 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6181 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6241 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6301 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6361 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6421 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6481 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6541 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6601 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6661 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6721 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6781 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6841 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6901 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6961 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7021 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7081 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7141 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7201 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
