LOCUS       sequence-251069-        7019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR (donor) Plasmid for Inserting PuroR-2A-GFP-AID into the 5' end 
            of the Mouse Srcap Gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7019)
  TITLE     Plasmids for generating AID/Degron Cell Lines in Human and Mouse
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7019)
  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: 
            "Unpublished"
COMMENT     Sequence Label: Mouse 5' Srcap AID GFP Puro
FEATURES             Location/Qualifiers
     source          1..7019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             953..1552
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MATEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     1452..1472
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1559..1612
                     /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             join(1622..1624,1625..2335)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1664..1685)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2272..2293
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2345..3025
                     /codon_start=1
                     /gene="Arabidopsis thaliana AXR3"
                     /product="auxin-responsive IAA17 protein"
                     /label=auxin-inducible degron
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="GSVELNLRETELCLGLPGGDTVAPVTGNKRGFSETVDLKLNLNNE
                     PANKEGSTTHDVVTFDSKEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGG
                     PEAAAFVKVSMDGAPYLRKIDLRMYKSYDELSNALSNMFSSFTMGKHGGEEGMIDFMNE
                     RKLMDLVNSWDYVPSYEDKDGDWMLVGDVPWPMFVDTCKRLRLMKGSDAIGLAPRAMEK
                     CKSRA"
     RBS             3932..3940
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(4158..4178)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4158..4176)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4197..4213)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4197..4213)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4210..4232)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4221..4237
                     /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(4245..4251,4252..4269,4270..4275))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4290..4311
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4428..4445)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4599..5187)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4679..4698)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5358..6149,6150..6218))
                     /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     5981..6000
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6219..6323)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6349..6804)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6486..6507)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6698..6717
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6931..6953
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6945..6962
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6946..6962
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6972..6991
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6972..6990
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 tgagataggg cttctattgt agcccaggct gtcctggaac tctttctgga gaccaggcta
       61 gcctcaaact aacagagatc cacttacctc tgtctcctga gtgctgaggt taaaggcatg
      121 taccatgaat gcttagcata tttattcttt tgaaatgaga tcttactttg tgtttgaggc
      181 tggtctctaa tgcctatgtt aaaacaatca tctccaatgt cccaaatagc tgtgaatatt
      241 ggcatataca gatatcaaga tatgtctgtc taagcaagtt cttacaggta tttgatacag
      301 agtaagtatc taagacatag ggatagatag tatgttgaga aagggtctcc gagtcaatgc
      361 atagccaaag atgaacttga acttctgatc ttcctttacc tctcaagctt ttggattgaa
      421 ggtgtgtgcc accctgctgg ttttatgcag ggttgaggat tgaacccttc ttgggtgcta
      481 ggccgagcca gctgagctac ctccatatcc ctgaaaatag ttcttaaccc cagcattggg
      541 agctgaggca ggaggattgt aagttccagg ccagtctgga tggtagaata agaccctaat
      601 tgtctcaaaa acaattattt taatagcctt taattttatt tctactcctt ttcttttcct
      661 ctctctctct ctctttcctt ctttctttct ttctttcttt ctttcttttt ttctttcttt
      721 ctttctttct ttttatccaa ctgcaggtac tccagaggct ggtgaacctg agcggaggct
      781 gggacgccct ggtgggcccc gggccctgga aggcaggtcc cggtggctgg tggcccagaa
      841 tgaggccagc tcccagcatg ccctgcagcc ggacaccagc cactcggcct gcagcaacgg
      901 tgataaccca gtcattcttc aggcatccag caaggagcct gggagtggga ccatggccac
      961 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     1021 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     1081 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     1141 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     1201 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     1261 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     1321 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     1381 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     1441 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     1501 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg ccagcggtga
     1561 aggcagaggc tctctcctca catgtgggga tgttgaagaa aatccaggtc ccatgtctag
     1621 cgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     1681 cgacgtaaac ggccacaagt tcagcgtgcg cggcgagggc gagggcgatg ccaccaacgg
     1741 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     1801 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     1861 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcacctt
     1921 caaggacgac ggcacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1981 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     2041 gctggagtac aacttcaaca gccacaacgt ctatatcacc gccgacaagc agaagaacgg
     2101 catcaaggcc aacttcaaga tccgccacaa cgtggaggac ggcagcgtgc agctcgccga
     2161 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2221 cctgagcacc cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2281 gctggagttc gtgaccgccg ccgggatcac tcacggcatg gacgagctgt acaagggagg
     2341 taccggcagc gtggaactga acctgcgcga gacagagctg tgcctgggac tgcctggcgg
     2401 cgatacagtg gctcctgtga ccggcaacaa gagaggcttc agcgagacag tggacctgaa
     2461 gctgaacctg aacaacgagc ccgccaacaa agagggcagc accacccacg acgtcgtgac
     2521 cttcgacagc aaagagaagt ccgcctgccc caaggacccc gccaagcctc ctgctaaagc
     2581 tcaggtcgtg ggatggcctc ccgtgcggag ctacagaaag aacgtgatgg tgtcctgcca
     2641 gaagtcctct ggcggccctg aggctgccgc cttcgtgaaa gtgtctatgg atggcgcccc
     2701 ttacctgaga aagatcgacc tgagaatgta caagagctac gacgagctga gcaacgccct
     2761 gagcaacatg ttcagcagct tcacaatggg caagcacggc ggcgaggaag gcatgatcga
     2821 cttcatgaac gagcggaagc tgatggacct cgtgaacagc tgggactacg tgcccagcta
     2881 cgaggacaag gacggcgact ggatgctcgt gggagatgtg ccctggccca tgttcgtgga
     2941 cacatgcaag agactgagac tgatgaaggg cagcgacgcc atcggcctgg cccccagagc
     3001 tatggaaaag tgcaagagca gagccggagg gtctggaggc tctggatccc agagcagccc
     3061 ctccactgct catcctcagc tcccaatcct acagacacag gtttgaaagt ggggaaggtt
     3121 cctatgatta tttctatgag ctaattcatg cctttgaaac aagtgatgtt gcaggtcttc
     3181 ttacccacct aagtttagac tctctttagt tctcagatag attggggagt aacatgtcaa
     3241 gcctaatttt ttcttcttta atttggtttt ctgagataag agtttcccca cctactgtct
     3301 tggaacttgc tctgtacacc aggctggcct tgaactcaga tccatttgtc tctgacttca
     3361 gatacttaac ttctacttaa cataataatg ccgatttcca agagtacata tcttgaaagg
     3421 tgtgctggcg cgtgccttta gcgtgccttt agtcacagca ctcaggcaga ggcaggcaga
     3481 tctctatgag ataaggccag taaacactat atagtgagag cctgtctttt tttttttttt
     3541 tttttttttt ggtttttcga gacagggttt ctctgtgtag ccctggctgt tctgaaactc
     3601 actttgtaga ctaggctggc ctcgaactca gaaatccgcc tgcctctgcc tcccaagtgc
     3661 tgggattaaa ggcgtgagcc aacactgcct agcctgagat cctatcttta aaaaaaagac
     3721 acatggactg gtgagatggc tcagtggttt agagcactca tggctcttcc agagttcaat
     3781 tcccagcaac cacatggtgg ctcacaacca tctgtaatag gattcagtga cctcttctga
     3841 tatgtctgaa gagagcacag tgtactcaca tacataaaaa tcttcaaaaa attattaaag
     3901 aaaagaaaaa agacacaggt ttttcttacc ataaggaggt ctaggctaag ccagaaggtg
     3961 gtggtgcatg cctttaatcc cagtatttag gaggcagagg cagttcaagg ccagcttgga
     4021 ctacagagca agttctagac agccaaggct acccagagac accctgacgt aaaataatga
     4081 aaaacgaaag aaagagaggt ctaggttagg ccgtaaatgg agcggccgcc accgcggtgg
     4141 agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     4201 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4261 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4321 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4381 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4441 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4501 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4561 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4621 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4681 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4741 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4801 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4861 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4921 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4981 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5041 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5101 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5161 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5221 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5281 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5341 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5401 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5461 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5521 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5581 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5641 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5701 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5761 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5821 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5881 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5941 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6001 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6061 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6121 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6181 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6241 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6301 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
     6361 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     6421 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     6481 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     6541 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     6601 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     6661 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     6721 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     6781 ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag
     6841 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     6901 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     6961 gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc cccctcgag
//
