LOCUS       sequence-321814-        3887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  homologous donor template for endogenous tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3887)
  AUTHORS   Gu B, Comerci CJ, McCarthy DG, Saurabh S, Moerner WE, Wysocka J
  TITLE     Opposing Effects of Cohesin and Transcription on CTCF Organization 
            Revealed by Super-resolution Imaging.
  JOURNAL   Mol Cell. 2020 Nov 19;80(4):699-711.e7. doi: 
            10.1016/j.molcel.2020.10.001. Epub 2020 Oct 21.
  PUBMED    33091336
REFERENCE   2  (bases 1 to 3887)
  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"; doi: 
            "10.1016/j.molcel.2020.10.001"; journalName: "Mol Cell"; date: 
            "2020-11-19- 19"; volume: "80"; issue: "4"; pages: "699-711.e7"
COMMENT     Sequence Label: pGEMT-aid-p2a-GFP
FEATURES             Location/Qualifiers
     source          1..3887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     162..183
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(301..319)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(302..319)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(337..353)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(337..353)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(350..372)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    361..377
                     /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(385..391,392..409,410..415))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    430..451
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(568..585)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(739..1327)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(819..838)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1498..2289,2290..2358))
                     /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     2121..2140
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2359..2463)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2541..2996)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2678..2699)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2890..2909
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3122..3144
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3136..3153
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3137..3153
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3160..3179
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3160..3178
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3209..3218
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3258..3389
                     /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(3399..3401,3402..3887,1..228)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3399..3401,3402..3887,1..225)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3441..3462)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3702..3721)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
ORIGIN
        1 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
       61 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
      121 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
      181 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaat cactatgcgg
      241 ccgcctgcag gtcgaccata tgggagagct cccaacgcgt tggatgcata gcttgagtat
      301 tctatagtgt cacctaaata gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
      361 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
      421 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
      481 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
      541 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
      601 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
      661 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
      721 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
      781 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
      841 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
      901 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
      961 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     1021 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     1081 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     1141 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     1201 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     1261 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     1321 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     1381 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     1441 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     1501 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     1561 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     1621 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     1681 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     1741 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     1801 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     1861 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     1921 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     1981 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     2041 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     2101 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     2161 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     2221 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     2281 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     2341 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     2401 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     2461 gcgcacattt ccccgaaaag tgccacctga tgcggtgtga aataccgcac agatgcgtaa
     2521 ggagaaaata ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     2581 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     2641 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     2701 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     2761 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     2821 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     2881 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     2941 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat
     3001 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     3061 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     3121 tcccagtcac gacgttgtaa aacgacggcc agtgaattgt aatacgactc actatagggc
     3181 gaattgggcc cgacgtcgca tgctcccggc cgccatggcc gcgggatgga ggaggtgggt
     3241 ctggaggtgg aggatctcct aaagatccag ccaaacctcc ggccaaggca caagttgtgg
     3301 gatggccacc ggtgagatca taccggaaga acgtgatggt ttcctgccaa aaatcaagcg
     3361 gtggcccgga ggcggcggcg ttcgtgaaga agcttgatgt gagcaagggc gaggagctgt
     3421 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     3481 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     3541 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     3601 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     3661 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     3721 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     3781 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     3841 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtg
//
