LOCUS       sequence-321819-        3891 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 3891)
  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 3891)
  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-GFP-p2a-aid
FEATURES             Location/Qualifiers
     source          1..3891
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(74..662)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(154..173)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(833..1624,1625..1693))
                     /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     1456..1475
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1694..1798)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1876..2331)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2013..2034)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2225..2244
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2457..2479
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2471..2488
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2472..2488
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2495..2514
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2495..2513
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2544..2553
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2565..2567,2568..2570,2571..3281)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2565..2567,2568..2570,2571..3281)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2610..2631)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2871..2890)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3218..3239
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3291..3422
                     /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"
     promoter        complement(3527..3545)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3528..3545)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3563..3579)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3563..3579)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3576..3598)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3587..3603
                     /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(3611..3617,3618..3635,3636..3641))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3656..3677
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3794..3811)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
       61 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
      121 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
      181 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
      241 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
      301 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
      361 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
      421 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
      481 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
      541 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      601 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
      661 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
      721 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
      781 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
      841 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
      901 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
      961 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     1021 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     1081 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     1141 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     1201 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     1261 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     1321 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     1381 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     1441 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     1501 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     1561 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     1621 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     1681 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     1741 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     1801 catttccccg aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     1861 aaataccgca tcaggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     1921 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     1981 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     2041 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     2101 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     2161 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     2221 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     2281 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tccattcgcc
     2341 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     2401 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     2461 gtcacgacgt tgtaaaacga cggccagtga attgtaatac gactcactat agggcgaatt
     2521 gggcccgacg tcgcatgctc ccggccgcca tggccgcggg atttatggtg agcaagggcg
     2581 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     2641 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     2701 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     2761 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     2821 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     2881 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     2941 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     3001 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     3061 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     3121 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     3181 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     3241 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggcagtgga cctaaagatc
     3301 cagccaaacc tccggccaag gcacaagttg tgggatggcc accggtgaga tcataccgga
     3361 agaacgtgat ggtttcctgc caaaaatcaa gcggtggccc ggaggcggcg gcgttcgtga
     3421 agggaggagg tgggtctgga ggtggaggat ctaatcacta gtgcggccgc ctgcaggtcg
     3481 accatatggg agagctccca acgcgttgga tgcatagctt gagtattcta tagtgtcacc
     3541 taaatagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     3601 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     3661 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     3721 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     3781 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3841 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat a
//
