LOCUS       sequence-191994-        5921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For amplification of a EGFP-miniAID for N-terminal tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5921)
  AUTHORS   Lambrus BG, Moyer TC, Holland AJ
  TITLE     Applying the auxin-inducible degradation system for rapid protein 
            depletion in mammalian cells.
  JOURNAL   Methods Cell Biol. 2018;144:107-135. doi: 
            10.1016/bs.mcb.2018.03.004. Epub 2018 Apr 5.
  PUBMED    29804665
REFERENCE   2  (bases 1 to 5921)
  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: "Methods 
            Cell Biol."; date: "2018"; volume: "144:107-135. doi"; pages: " 
            10.1016/bs.mcb.2018.03.004. Epub 2018 Apr 5"
COMMENT     Sequence Label: pcDNA5/FRT EGFP-miniAID
FEATURES             Location/Qualifiers
     source          1..5921
                     /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"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(926..928,929..1639)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(926..928,929..1639)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(968..989)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1229..1248)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1576..1597
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1873..1890)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1879..2103
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2236..2255)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2387..2434
                     /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             2442..3464
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2442..3464
                     /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    3594..3715
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3631..3650)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3685..3704
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3764..3780)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3764..3780)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3777..3799)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3788..3804
                     /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(3812..3818,3819..3836,3837..3842))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3857..3878
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3995..4012)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4166..4754)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4246..4265)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4925..5716,5717..5785))
                     /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     5548..5567
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5786..5890)
                     /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 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 ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttatgg gtaccgtgag caagggcgag gagctgttca ccggggtggt
      961 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1021 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1081 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     1141 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1201 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1261 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1321 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     1381 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     1441 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1501 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     1561 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     1621 catggacgag ctgtacaagg ctagcggcgg atctgactct aaagagaagt cagcttgtcc
     1681 taaggaccct gccaagccac ctgcgaaggc ccaagtagtc ggttggccac ccgtgcgcag
     1741 ctataggaag aacgtgatgg tgtcctgtca gaagtctagc ggggggcccg agggatccgc
     1801 tgctgccgcc ggcggatctg atatctaggc ggccgctcga gtctagaggg cccgtttaaa
     1861 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     1921 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     1981 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2041 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2101 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     2161 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2221 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2281 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2341 acctcgaccc caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc
     2401 gaagttccta ttctctagaa agtataggaa cttccttggc caaaaagcct gaactcaccg
     2461 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc
     2521 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc
     2581 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg
     2641 catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga
     2701 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac
     2761 tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta
     2821 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc
     2881 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg
     2941 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact
     3001 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca
     3061 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg
     3121 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct
     3181 acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc
     3241 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt
     3301 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac
     3361 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata
     3421 gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgta ctacgagatt
     3481 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     3541 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     3601 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     3661 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     3721 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     3781 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     3841 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     3901 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3961 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4021 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4081 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4141 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4201 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4261 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4321 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4381 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4441 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4501 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4561 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     4621 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4681 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4741 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4801 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4861 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4921 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4981 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5041 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5101 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5161 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5221 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5281 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5341 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5401 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5461 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5521 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5581 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5641 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5701 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5761 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5821 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5881 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
