LOCUS       sequence-487564-        6146 bp    DNA     circular SYN 01-OCT-2025
DEFINITION  Expression of Ring2-FLAG-AID.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6146)
  TITLE     Hengbin lab plasmids 2025
REFERENCE   2  (bases 1 to 6146)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 1, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: Ring2-FLAG-AID-mClover
FEATURES             Location/Qualifiers
     source          1..6146
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     177..196
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     430..447
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    564..585
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(600..605,606..623,624..630)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    638..654
                     /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)."
     primer_bind     643..665
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     662..678
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     662..678
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     697..717
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        699..717
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             1585..1608
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1609..1812
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             join(1819..1821,1822..1824,1825..2535)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1864..1885)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2472..2493
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2536..2589
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2590..3189
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2590..2609)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3086..3106
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4030..4049)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4031..4049)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4059..4076)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4059..4075)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4068..4090)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4217..4672
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4304..4323)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4514..4535
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4698..4802
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4803..4871,4872..5663)
                     /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     complement(5021..5040)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5834..6146,1..276)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
       61 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
      121 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
      181 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
      241 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
      301 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
      361 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
      421 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
      481 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
      541 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
      601 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
      661 acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa
      721 caaaagctgg ccgttctggt cattagggtt tggggggtat gttccttttt ttccccttag
      781 tactttgtgt atttctgttt gatttttgtc actgtgtatc tcattttata gaaggtaagg
      841 taggtaaagc ttttgtgacc tgacagtaag atattgagta tttactttcc cctcttctgt
      901 acctatcaat aagaaattta gattgtagag cagtaaattt ctgatattat tttgtcaata
      961 attgagacat tgccatttta cttagttttt aagaaatgta ttagttccat aatttaagta
     1021 catttttctc attggcatat tctagaaatg aaatttttaa tgatttattc ttctagatac
     1081 ataaagactt ctggtaacgc cactgttgat cacttatcca agtatctggc tgtgaggtta
     1141 gctttagaag aacttcgaag caaaggtgaa tcaaaccaga tgaaccttga tacagccagt
     1201 gagaagcagt ataccattta tatagcaaca gccagtggcc agttcactgt gagtatttaa
     1261 aataatagac tgttgaaact gggagcacac tgaccaacta actgagtggc aagtggtggg
     1321 gtagaatcta ataaataatg taaaaaaccg taacagtgtt aagtgacaag tagttattcc
     1381 taagatttga aaaaaaaaga ttctgtggac ttccatttta gtttcatttc attattattg
     1441 tggtttgtgc agttttcata attttttctt tcttttttgt tttaggtatt aaatggctct
     1501 ttttctttgg aattggtcag tgagaaatac tggaaagtga acaaacccat ggagctgtac
     1561 tatgctccta caaaggagca caaagattac aaagacgatg acgataagaa ggagaagagt
     1621 gcttgtccta aagatccagc caaacctccg gccaaggcac aagttgtggg atggccaccg
     1681 gtgagatcat accggaagaa cgtgatggtt tcctgccaaa aatcaagcgg tggcccggag
     1741 gcggcggcgt tcgtgaaggt atcaatggac ggagcaccgt acttgaggaa aatcgatttg
     1801 aggatgtata aagctagcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1861 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtccg cggcgagggc
     1921 gagggcgatg ccaccaacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1981 cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcgtggcctg cttcagccgc
     2041 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgccaga aggctacgtc
     2101 caggagcgca ccatctcttt caaggacgac ggtacctaca agacccgcgc cgaggtgaag
     2161 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2221 ggcaacatcc tggggcacaa gctggagtac aacttcaaca gccacaacgt ctatatcacg
     2281 gccgacaagc agaagaacgg catcaaggct aacttcaaga tccgccacaa cgttgaggac
     2341 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2401 ctgctgcccg acaaccacta cctgagccat cagtccaagc tgagcaaaga ccccaacgag
     2461 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggattac acatggcatg
     2521 gacgagctgt acaaggaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat
     2581 cctggcccaa tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     2641 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     2701 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     2761 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     2821 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     2881 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     2941 cccaaggagc ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag
     3001 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     3061 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     3121 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     3181 ggtgcctgag cctttaaaaa ccaattctga gactgaactt ttttatagcc tatttcttta
     3241 atattaaaga tgtactggca ttacttttat ggacagatct tggatatgtt gttcaatttt
     3301 ctttctgagc cagactagtt tacgctattc aaatcttttc ccctttattt aagatttcct
     3361 ttttggaagg gactgcaatt attcagtatt tttttctttc ctttaaaaaa atatatctga
     3421 agtttcttgt gttttttttt ttccccacaa agtgtgtttc cacttggagc accattttga
     3481 cccaggaatt tttcatagtt tctgtattct tataagattc agttggctgt ccttttcctg
     3541 ctcccctcaa aagattttta gtcatacaga atgttaaata ttatgtattc tgactttttt
     3601 tttcccccgg agtcttgtat atttatagtt ttctatataa actgtagtat cttcatgaag
     3661 acccaaggct caaatttact gtccttaaaa acaattctca taggattatt cttttcatgg
     3721 tattttcttc cataatatct cattttaaaa agaagttctt tatgaactta gtgtccattg
     3781 tcatgcaatg tttttttttc cattcttttt ccctgtaatt ttggaatttc tggtcctggg
     3841 aagaatcaaa caaaatctta agttctatga gaacttggtt cattgacata ttctgctgaa
     3901 gaaagaaaaa ttaaattggt agtaaaatat agtcttcaag tatacgtttg agagtgcttt
     3961 tttttgtatt agttctgctg tcacttcatt tcctgtatta tatgtgatgt ttttcccccg
     4021 gccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     4081 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     4141 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     4201 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4261 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4321 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4381 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     4441 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4501 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     4561 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     4621 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     4681 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4741 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4801 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4861 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4921 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4981 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5041 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5101 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5161 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5221 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5281 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5341 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5401 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     5461 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     5521 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     5581 acacgacggg gagtcaggca actatggatg aacgaaatag acagatagct gagataggtg
     5641 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     5701 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5761 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5821 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5881 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5941 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6001 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6061 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6121 agttaccgga taaggcgcag cggtcg
//
