LOCUS       sequence-438225-        6899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repair template to endogenously tag WAPL with Hygro-P2A-HA-FKBP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6899)
  AUTHORS   Batty P, Langer CC, Takacs Z, Tang W, Blaukopf C, Peters JM, Gerlich
            DW
  TITLE     Cohesin-mediated DNA loop extrusion resolves sister chromatids in G2
            phase.
  JOURNAL   EMBO J. 2023 Aug 15;42(16):e113475. doi: 10.15252/embj.2023113475. 
            Epub 2023 Jun 26.
  PUBMED    37357575
REFERENCE   2  (bases 1 to 6899)
  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: "EMBO J."; 
            date: "2023-08-15"; volume: "42(16):e113475. doi"; pages: " 
            10.15252/embj.2023113475. Epub 2023 Jun 26"
COMMENT     Sequence Label: Hygro-P2A-HA-FKBP-WAPL
FEATURES             Location/Qualifiers
     source          1..6899
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    29..45
                     /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)."
     primer_bind     34..56
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     53..69
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     53..69
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(847..864)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(874..891)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(940..1260)
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             complement(1282..1308)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(1312..1338)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             complement(1312..1338)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(1339..1395)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(1405..2424)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(1405..2424)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     primer_bind     complement(3180..3199)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3181..3199)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3206..3223)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3206..3222)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3215..3237)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3363..3791
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3450..3469)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3660..3681
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             4135..4929
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4189..4208)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4799..4818
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(4947..5015,5016..5807)
                     /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     complement(5165..5184)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5978..6566
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6467..6486
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6720..6737
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6854..6875
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6890..6895,6896..6899,1..14,15..21)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
       61 agctatgacc atgattacgc caagcttggt accgagctcg gatccactag taacggccgc
      121 cagtgtgctg gaattctgca gatataaatg ttaacatcca agttgtttat ttcaacctcc
      181 aactggtttc tttaaagttc ttaaatgtct tttacaaaac tatcaatggt ccccatgtat
      241 cataagtgca gaaagaaaac acaatatata ttttagaatt taaatcattt aaataaattg
      301 gttgcacaaa gctctcttgg cggagaataa agaaaatcaa aacttacctg aagttattaa
      361 tttattacag ctacttatgc ttgcatcatc ttctacaatt cggtttgtgc tcttttcttt
      421 cccaagtaaa gtgtcctcca aagggaagca tttatcagaa acgacagtgt cttcaacgac
      481 cacatgacta attttgctat tagcttcaag tactgaagtg acctcttcca actgagcagc
      541 ttcactagat tctgaataag aggaacactt aacctgggct aaattctttg aagaaactgg
      601 tagagactca tcatcactgt caaatccaaa aggatcacca gtactttctt cttccacttt
      661 aggtttcttc ggaatttctt ggatatctgg tttgaaattg ggcctcttct gccctaattt
      721 agccataaat gtggtctctc cccacttagt acttagcgtt gtgcgtttgt tcgaaaacac
      781 ttcatcgaat tttgaactgc catttccacc ttttctactg taagttttgc caaatcgcga
      841 tgtatggctg ccgcgcggca ccaggccgct gctatgatgg tgatgatggt gactgctcgt
      901 cgactgcaga attcgaagct tgagctcgag gtctgagtat tccagtttta gaagctccac
      961 atcgaagacg agagtggcat gtggtgggat gatgcctggg tgcccagtgg caccataggc
     1021 ataatctggt gatatagtca gtttggctct ctgacccaca ctcatctggg caaccccttc
     1081 ttcccagcct cggatcacct cctgcttgcc tagcataaac ttaaaaggct tgtttctgtc
     1141 ccgggaggaa tcaactttct ttccatcttc aagcatcccg gtgtagtgca ccacgcaggt
     1201 ctggccgcgc ttggggaagg tgcgcccgtc tcctggggaa atggtttcca cctgcactcc
     1261 tccacctcca cctccacgcg ttgcatagtc cgggacatca tacggatagc cggcgtagtc
     1321 gggcacgtcg taggggtaag gtccagggtt ctcctccacg tctccagcct gcttcagcag
     1381 gctgaagtta gtagctccgc ttccttcctt tgccctcgga cgagtgctgg ggcgtcggtt
     1441 tccactatcg gcgagtactt ctacacagcc atcggtccag acggccgcgc ttctgcgggc
     1501 gatttgtgta cgcccgacag tcccggctcc ggatcggacg attgcgtcgc atcgaccctg
     1561 cgcccaagct gcatcatcga aattgccgtc aaccaagctc tgatagagtt ggtcaagacc
     1621 aatgcggagc atatacgccc ggagccgcgg cgatcctgca agctccggat gcctccgctc
     1681 gaagtagcgc gtctgctgct ccatacaagc caaccacggc ctccagaaga agatgttggc
     1741 gacctcgtat tgggaatccc cgaacatcgc ctcgctccag tcaatgaccg ctgttatgcg
     1801 gccattgtcc gtcaggacat tgttggagcc gaaatccgcg tgcacgaggt gccggacttc
     1861 ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc tgcgcgacgg acgcactgac
     1921 ggtgtcgtcc atcacagttt gccagtgata cacatgggga tcagcaatcg cgcatatgaa
     1981 atcacgccat gtagtgtatt gaccgattcc ttgcggtccg aatgggccga acccgctcgt
     2041 ctggctaaga tcggccgcag cgatcgcatc catggcctcc gcgaccggct gcagaacagc
     2101 gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc tgtgcacggc gggagatgca
     2161 ataggtcagg ctctcgctga attccccaat gtcaagcact tccggaatcg ggagcgcggc
     2221 cgatgcaaag tgccgataaa cataacgatc tttgtagaaa ccatcggcgc agctatttac
     2281 ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa gcacgagatt cttcgccctc
     2341 cgagagctgc atcaggtcgg agacgctgtc gaacttttcg atcagaaact tctcgacaga
     2401 cgtcgcggtg agttcaggct tcatacgcgt tttgacagca gtttcatatt ctctgtgaaa
     2461 aaaaatttaa gaaaacatat aatcatcaaa tacaagtgtt aaacagtgca tataaaaata
     2521 aaaacctagg gattcttccc tcatgacttt tgacactcac cacacagact tttaaataaa
     2581 caaatatgag gaaggggtat aaagaaaggc tgatttatta ccctacatgc ttccatgtca
     2641 attttcctcc acaatgagta catattccta cgctacttac aaagtaaaaa aacttaatga
     2701 ttaaccaact tataatacga tatccataaa gctaacttaa aatgaagcat attaagttcc
     2761 aaatgaatta atttactatt cttataaaca tcatttttac cttaaaaaat tatctgtata
     2821 gaggctggga ggccgaggca ggcagctcac ttgaggcagg agtttgagac cagcctgccc
     2881 aacacggtga aaccccatct ctactaaaaa tacaaaaatt agctgggagt ggtggcacgc
     2941 acctgtaatc ccagctactt gagaagctgg agcagaagaa ccgcttgaac tcaggagatg
     3001 gaggttgcag tgatataatg ataacctgta tagggtaatt acttatataa aatcttaagc
     3061 acagaatgtt agtttttttc agggcctcag aaatatccaa tacttgatta aatttagttc
     3121 tttgaaatta tccatcacac tggcggccgc tcgagcatgc atctagaggg cccaattcgc
     3181 cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     3241 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     3301 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     3361 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3421 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3481 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3541 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3601 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3661 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3721 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3781 taacgcgaat tttaacaaaa ttcagggcgc aagggctgct aaaggaagcg gaacacgtag
     3841 aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg ggctatctgg
     3901 acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct tacatggcga
     3961 tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc
     4021 tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc
     4081 tgatggcgca ggggatcaag atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4141 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4201 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4261 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     4321 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4381 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4441 ctgtcatccc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4501 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     4561 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     4621 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     4681 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     4741 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4801 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4861 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4921 ttcttctgaa ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     4981 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5041 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5101 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5161 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5221 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5281 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5341 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5401 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5461 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     5521 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     5581 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     5641 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     5701 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     5761 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     5821 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     5881 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5941 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6001 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6061 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6121 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6181 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6241 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6301 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6361 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6421 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6481 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6541 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6601 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     6661 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     6721 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     6781 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     6841 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttt
//
