LOCUS       sequence-385200-        6955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeting vector to introduce AID-eGFP tags in C-terminal of the 
            human RPB1 gene. Blasticidin selection (2A-fusion). Auxin-inducible 
            degron system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6955)
  AUTHORS   Valton AL, Venev SV, Mair B, Khokhar ES, Tong AHY, Usaj M, Chan K, 
            Pai AA, Moffat J, Dekker J
  TITLE     A cohesin traffic pattern genetically linked to gene regulation.
  JOURNAL   Nat Struct Mol Biol. 2022 Dec 8. doi: 10.1038/s41594-022-00890-9.
  PUBMED    36482254
REFERENCE   2  (bases 1 to 6955)
  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: "Nat Struct 
            Mol Biol. 2022 Dec 8. doi: 10.1038/s41594-022-00890-9."
COMMENT     Sequence Label: 221-RPB1-AID-eGFP-2A-blasticidin
FEATURES             Location/Qualifiers
     source          1..6955
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(36..55)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(37..55)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(60..76)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(60..76)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(178..197)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             189..998
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(256..275)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1145..1733
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1634..1653
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1887..1904
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2063..2090)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2182..2268)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2317..2339
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2331..2348
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2332..2348
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4067..4198
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Morawska and Ulrich, 
                     2013)"
                     /label=AID*
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="PKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVK"
     CDS             join(4208..4210,4211..4921)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4208..4210,4211..4921)
                     /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(4511..4530)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4858..4879
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4931..4984
                     /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"
ORIGIN
        1 gccctcacat cattatttgc catccagctg atatccccta tagtgagtcg tattacatgg
       61 tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac
      121 aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa
      181 ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg
      241 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
      301 tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
      361 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
      421 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
      481 cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
      541 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
      601 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
      661 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
      721 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
      781 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
      841 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
      901 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
      961 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt
     1021 aacactggca gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc
     1081 ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1141 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1201 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1261 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     1321 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1381 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     1441 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     1501 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     1561 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     1621 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     1681 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     1741 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     1801 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     1861 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     1921 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     1981 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatacgcgt accgctagcc
     2041 aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttagtttg
     2101 atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac
     2161 gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca
     2221 gataaaacga aaggcccagt cttccgactg agcctttcgt tttatttgat gcctggcagt
     2281 tccctactct cgcgttaacg ctagcatgga tgttttccca gtcacgacgt tgtaaaacga
     2341 cggccagtct taagctgttc agcacccagt cccatgggtg gaatctctcc tgccatgaca
     2401 ccttggaacc agggtgcaac ccctgcctat ggcgcctggt cccccagtgt tggtgagtaa
     2461 cctgatccag gaagagggcc ctccaggtgg ttcagaggtg gataagctgg gatcgtgaac
     2521 ggtagccatt gttggaactc tgaaggccgc agagggctag tgaaaggtgg ggcagggaga
     2581 attgggggcc tttggtctcc cgctctggcc ttaacttgct tctctcactc tatagggagt
     2641 ggaatgaccc caggggcagc cggcttctct cccagtgctg cgtcagatgc cagcggcttc
     2701 agcccaggtt actcccctgc ctggtctccc acaccgggct ccccggggtc cccaggtccc
     2761 tcaagcccct acatcccttc accaggtgag ttgtcgccat ctgtcaggcc aggtttgttt
     2821 cctccacagt tctgggtgag tctgtccatt gctcatctcc tttgttggct gcttccccac
     2881 aggtggtgcc atgtctccca gctactcgcc aacgtcacct gcctacgagc cccgctctcc
     2941 tgggggctac acaccccaga gtccctctta ttcccccact tcaccctcct actcccctac
     3001 ctctccatcc tattctccaa ccagtcccaa ctatagtccc acatcaccca gctattcgcc
     3061 aacgtcaccc agctactcac cgacctctcc cagctactca cccacctctc ccagctactc
     3121 gcccacctct cccagctact cgcccacctc tcccagctac tcacccactt cccctagcta
     3181 ctcgcccact tcccctagct actcgccgac gtctcccagc tactcgccga catctcccag
     3241 ctactcgcca acttcaccca gctattctcc cacttctccc agctactcac ctacctctcc
     3301 aagctattca cccacctccc ccagctactc acccacttcc ccaagttact cacccaccag
     3361 cccgaactat tctccaacca gtcccaatta caccccaaca tcacccagct acagcccgac
     3421 atcacccagc tattcaccta ctagtcccaa ctacacacct accagcccta actacagccc
     3481 aacctctcca agctactctc caacatcacc cagctattcc ccgacctcac caagttactc
     3541 cccttccagc ccacgataca caccacagtc tccaacctat accccaagct cacccagcta
     3601 cagccccagc tcgcccagct acagcccaac ctcacccaag tacaccccaa ccagtccttc
     3661 ttacagtccc agctccccag agtatacccc aacctctccc aagtactcac ctaccagtcc
     3721 caaatattca cccacctctc ccaagtactc gcctaccagt cccacctatt cacccaccac
     3781 cccaaaatac tccccaacat ctcctactta ttccccaacc tctccagtct acaccccaac
     3841 ctctcccaag tactcaccta ctagccccac ttactcgccc acttccccca agtactcgcc
     3901 caccagcccc acctactcgc ccacctcccc caaaggctca acctactctc ccacttcccc
     3961 tggttactcg cccaccagcc ccacctacag tctcacaagc ccggctatca gcccggatga
     4021 cagtgacgag gagaacgggg gagcaggagg cgtcgacggt atcgatccta aagatccagc
     4081 caaacctccg gccaaggcac aagttgtggg atggccaccg gtgagatcat accggaagaa
     4141 cgtgatggtt tcctgccaaa aatcaagcgg tggcccggag gcggcggcgt tcgtgaagaa
     4201 gcttgatgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     4261 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     4321 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     4381 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     4441 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     4501 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     4561 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     4621 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     4681 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     4741 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     4801 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     4861 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     4921 gggcagtgga gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg
     4981 cccaatgaaa accttcaaca tctctcagca ggatctggag ctggtggagg tcgccactga
     5041 gaagatcacc atgctctatg aggacaacaa gcaccatgtc ggggcggcca tcaggaccaa
     5101 gactggggag atcatctctg ctgtccacat tgaagcctac attggcaggg tcactgtctg
     5161 tgctgaagcc attgccattg ggtctgctgt gagcaacggg cagaaggact ttgacaccat
     5221 tgtggctgtc aggcacccct actctgatga ggtggacaga tccatcaggg tggtcagccc
     5281 ctgtggcatg tgtagagagc tgatctctga ctatgctcct gactgctttg tgctcattga
     5341 gatgaatggc aagctggtca aaaccaccat tgaggaactc atccccctca agtacaccag
     5401 gaactaatga gggcacgtgg ggtgcggcag cgggctaggg cccagggcag cttgcccgtg
     5461 ctgccgtgca gttcttgcct ccctcacggg gcgtcacccc cagcccagct ccgttgtaca
     5521 taaatgcctt gtggcagagc tcccggtgaa cttctggatc ccgtttctga tgcagattct
     5581 tgtcttgttc tccacttgtg ctgttagaac tcactggccc agtggtgttc tcactcctac
     5641 cccacccacc ccctgcctgt ccccaaattg aagatccttc cttgcctgtg gcttgatgcg
     5701 gggcgggtaa agggtatttt aacttagggg tagttcctgc tgtgagtggt tacagctgat
     5761 cctcgggaag aacaaagcta aagctgcctt ttgtctgtta ttttattttt ttgaagttta
     5821 aataaagttt actaattttg accaaaagta tgtacatcga gtgcttcatt ggggttcagg
     5881 agacccccag gcccctgcct cctgcctggg ctgctgcttg atctgttagg gggaggggac
     5941 caggaaaatg ggagggtacc ttacagtcag cccacacaca gcccctgctc tgtgccaggc
     6001 actgctctgt actgagtttg ttgtagtcac aacagatgca gctcttgtcc tctattaagg
     6061 tgatattgac cataacagct ttttaacttg ttttactgat tgtcataaat aatttggaaa
     6121 agtgtagaaa agtataatgc agagggagaa aaatgatcac ccataatccc accactagga
     6181 ggcatacttt ctttggtatt ttttcctata cttttcttga gtcactaaca agtgaacaaa
     6241 aatgtacttt gattttggcc tcaaaccaag aatgaaaaga ttatatagat gttgtaatac
     6301 agaagctgaa gtaaaaggac ttgggggtaa tacgctagtt ttagccggct atttttcccc
     6361 ctttcattag caccttaatg tggtatcaat gttctacatc ctctgcaagt catttctgat
     6421 ttacctgagg tatttttatt tttattttat ttttttgaga cagtctcgct ctgttgccca
     6481 ggctggagtg cagtggcgca gtctcggctc actacaacct ctgcctcttg ggttcaagca
     6541 attctcactg cctcagcctt ccaagtagct gcgattgcag atgtccgtca ccatgcccag
     6601 ctactttggt atttttaata gagaagggat ttcacagtgt tggccaggct ggtctcaaac
     6661 tcctgatttc aggtgatctg tccactttgg cctcccaaag tgctgggatt acaggcatga
     6721 gccactgcac ctggcctgtt atctagaaat gtctattttg ccttcattcc taaagaatat
     6781 ctttgctaga gataggattc tggtgtgaca gattcatttt ttgtttttag cacttttttt
     6841 ttttttttag gagcagaggt ttaatatgca aaagatagag aaaagagaac agctcgctct
     6901 cttgtggggg agaggggctt ctgaaaggga agtccctaag atgtcctatc ttccg
//
