LOCUS       sequence-423991-        6064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For tagging the C-term of Top2A with Venus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6064)
  AUTHORS   Hildebrand EM, Polovnikov K, Dekker B, Liu Y, Lafontaine DL, Fox AN,
            Li Y, Venev SV, Mirny LA, Dekker J
  TITLE     Mitotic chromosomes are self-entangled and disentangle through a 
            topoisomerase-II-dependent two-stage exit from mitosis.
  JOURNAL   Mol Cell. 2024 Mar 13:S1097-2765(24)00144-8. doi: 
            10.1016/j.molcel.2024.02.025.
  PUBMED    38521067
REFERENCE   2  (bases 1 to 6064)
  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: "Mol Cell. 
            2024 Mar 13:S1097-2765(24)00144-8. doi: 
            10.1016/j.molcel.2024.02.025."
COMMENT     Sequence Label: pUC19-Top2A-5X Gly-Venus-T2A-Hygro
FEATURES             Location/Qualifiers
     source          1..6064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     509..528
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(747..851)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     919..937
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(975..997)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1097..1116
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1310..1332
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1324..1341
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1325..1341
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(2158..2160,2161..2163,2164..2877)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2203..2224)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2464..2483)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2811..2832
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2878..2931
                     /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"
     primer_bind     complement(4789..4805)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4789..4805)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4802..4824)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4813..4829
                     /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(4837..4843,4844..4861,4862..4867))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4882..4903
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5020..5037)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5191..5779)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5271..5290)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5950..6064,1..677,678..746))
                     /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"
ORIGIN
        1 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
       61 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
      121 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
      181 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
      241 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
      301 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
      361 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
      421 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
      481 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
      541 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
      601 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
      661 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
      721 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
      781 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
      841 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
      901 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
      961 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     1021 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     1081 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     1141 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg
     1201 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     1261 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     1321 cgttgtaaaa cgacggccag tgagctactc aggaggctga ggcaggagaa tcgcttgaac
     1381 ccaggaggca gaggttgcag tgagccgaga taacaccact gcactccagc ctggacgata
     1441 gagtgagacc ccatctcaaa aaaagagcag ctgtgacaaa tgcctgtatt gaattgcagg
     1501 tcagtcttcc acctccacta ccggtgccaa aaaaagggct gccccaaaag gaactaaaag
     1561 ggatccagct ttgaattctg gtgtctctca aaagcctgat cctgccaaaa ccaagaatcg
     1621 ccgcaaaagg aagccatcca cttctgatga ttctgactct aattttgaga aaattgtttc
     1681 gaaagcagtc acaagcaagg tgagtgttga tcctagtcag tccttttgct gtagatgttc
     1741 tgaaacacgt aactaagcca ttgctcttaa aaatttggca tatctttaag aaaattaact
     1801 ctcatattct gttagctttt actgtacata tttagtttta acaaagttaa atatgccact
     1861 tatttgggca atggaagagt tggccttaga tctgcttctt attacttggt agaaaataga
     1921 aaactccttg aatatagtgt cttgatacat ttttttacat tacaattatg ttgtcagatt
     1981 tacaatgtgc aagttacctg ggcttttctc ttttagaaat ccaaggggga gagtgatgac
     2041 ttccatatgg actttgactc agctgtggct cctcgggcaa aatctgtacg ggcaaagaaa
     2101 cctataaagt atctcgaaga gagagatgaa gatgatctgt ttggtggtgg tggtggtatg
     2161 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     2221 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     2281 aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     2341 gtgaccaccc tgggctacgg cctgcagtgc ttcgcccgct accccgacca catgaagcag
     2401 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2461 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2521 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2581 ctggagtaca actacaacag ccacaacgtc tatatcaccg ccgacaagca gaagaacggc
     2641 atcaaggcca acttcaagat ccgccacaac atcgaggacg gcggcgtgca gctcgccgac
     2701 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2761 ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2821 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagag
     2881 ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc aaccatgaaa
     2941 aagcctgaac tcaccgctac ctctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     3001 tccgacctga tgcagctctc cgatggcgaa gaatctcggg ctttcagctt cgatgtggga
     3061 gggcgtggat atgtcctgcg ggtgaatagc tgcgccgatg gtttctacaa agatcgctat
     3121 gtttatcggc actttgcatc cgccgctctc cctattcccg aagtgcttga cattggggag
     3181 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac cttgcaagac
     3241 ctgcctgaaa ccgaactgcc cgctgttctc cagcccgtcg ccgaggccat ggatgccatc
     3301 gctgccgccg atcttagcca gaccagcggg ttcggcccat tcggacctca aggaatcggt
     3361 caatacacta catggcgcga tttcatctgc gctattgctg atccccatgt gtatcactgg
     3421 caaactgtga tggacgacac cgtcagtgcc tccgtcgccc aggctctcga tgagctgatg
     3481 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgccgattt cggctccaac
     3541 aatgtcctga ccgacaatgg ccgcataaca gccgtcattg actggagcga ggccatgttc
     3601 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccctggtt ggcttgtatg
     3661 gagcagcaga cccgctactt cgagcggagg catcccgagc ttgcaggatc tcctcggctc
     3721 cgggcttata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     3781 ttcgatgatg cagcttgggc tcagggtcgc tgcgacgcaa tcgtccggtc cggagccggg
     3841 actgtcgggc gtacacaaat cgcccgcaga agcgctgccg tctggaccga tggctgtgtg
     3901 gaagtgctcg ccgatagtgg ataaaatgtg aggcgattat tttaagtaat tatcttacca
     3961 agcccaagac tggttttaaa gttacctgaa gctcttaact tcctcccctc tgaatttagt
     4021 ttggggaagg tgtttttagt acaagacatc aaagtgaagt aaagcccaag tgttctttag
     4081 ctttttataa tactgtctaa atagtgacca tctcatgggc attgttttct tctctgcttt
     4141 gtctgtgttt tgagtctgct ttcttttgtc tttaaaacct gatttttaag ttcttctgaa
     4201 ctgtagaaat agctatctga tcacttcagc gtaaagcagt gtgtttatta accatccact
     4261 aagctaaaac tagagcagtt tgatttaaaa gtgtcactct tcctcctttt ctactttcag
     4321 tagatatgag atagagcata attatctgtt ttatcttagt tttatacata atttaccatc
     4381 agatagaact ttatggttct agtacagata ctctactaca ctcagcctct tatgtgccaa
     4441 gtttttcttt aagcaatgag aaattgctca tgttcttcat cttctcaaat catcagaggc
     4501 cgaagaaaaa cactttggct gtgtctataa cttgacacag tcaatagaat gaagaaaatt
     4561 agagtagtta tgtgattatt tcagctcttg acctgtcccc tctggctgcc tctgagtctg
     4621 aatctcccaa agagagaaac caatttctaa gaggactgga ttgcagaaga ctcggggaca
     4681 acatttgatc caagatctta aatgttatat tgataaccat gctccgagct cggtacccgg
     4741 ggatcctcta gagtcgacct gcaggcatgc aagcttggcg taatcatggt catagctgtt
     4801 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4861 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4921 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4981 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5041 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5101 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5161 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5221 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5281 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5341 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5401 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5461 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5521 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5581 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5641 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5701 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5761 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5821 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5881 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5941 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6001 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6061 atct
//
