LOCUS       sequence-441366-        5932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DONOR homology arm-containing repair template used to insert 
            Auxin-Inducible Degron tags, mClover, and Hygromycin resistance 
            markers to the N-terminus of LRR1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5932)
  AUTHORS   Jones RM, Ruiz JH, Scaramuzza S, Nath S, Liu C, Henklewska M, 
            Natsume T, Bristow RG, Romero F, Kanemaki MT, Gambus A
  TITLE     Characterizing replisome disassembly in human cells.
  JOURNAL   iScience. 2024 Jun 12;27(7):110260. doi: 10.1016/j.isci.2024.110260.
            eCollection 2024 Jul 19.
  PUBMED    39055910
REFERENCE   2  (bases 1 to 5932)
  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: "iScience.";
            date: "2024-06-12"; volume: "27(7):110260. doi"; pages: " 
            10.1016/j.isci.2024.110260. eCollection 2024 Jul 19"
COMMENT     Sequence Label: pBS-HYGRO-P2A-mAID-mCLOVER-LRR1
FEATURES             Location/Qualifiers
     source          1..5932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1119..2141
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1119..2141
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2154..2210
                     /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             2217..2420
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             join(2427..2429,2430..2432,2433..3143)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2472..2493)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3080..3101
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3744..3764)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3744..3762)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3783..3799)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3783..3799)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        complement(join(3831..3837,3838..3855,3856..3861))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3876..3897
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4014..4031)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4185..4773)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4265..4284)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4944..5735,5736..5804))
                     /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     5567..5586
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5805..5909)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcag
      661 ttctaagtgc ctttccctgt aacgcagagg cacacctgcc ttccctcggg aaacagcggc
      721 ccgacgtgcg ccggcaacgc gtaaagcgtc aacagctgaa accctcggaa tcctccccga
      781 acagaaacaa ccaccgctgc cagctgcgcg ctcgggggaa aagacgttgc gcccccgccg
      841 actgccggtt tcccgggcgc gagcccggat ccaggtggtc agtcccggta cgcaaccacg
      901 gcgagaaccc ggccctgcta agggagaagg gaagccgttt cccgcgggct tcatacagac
      961 acggtgctaa gacagcccga gtcgcggacc agtccaacag gcaggcagat cagtgggcag
     1021 cccgcgtgcg cgaggacccc gatggcggcg ccgggtggcg ggaaggagga agtttcaaag
     1081 ccagcttgac gtggttgtgg ccgttggata tcgccaccat gaaaaagcct gaactcaccg
     1141 cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc
     1201 tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc
     1261 tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg
     1321 catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga
     1381 cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac
     1441 tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta
     1501 gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc
     1561 gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg
     1621 acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact
     1681 gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca
     1741 atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg
     1801 aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct
     1861 acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc
     1921 gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt
     1981 gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac
     2041 aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata
     2101 gtggaaaccg acgccccagc actcgtccga gggcaaagga atcaggaagc ggagctacta
     2161 acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct agatctaagg
     2221 agaagagtgc ttgtcctaaa gatccagcca aacctccggc caaggcacaa gttgtgggat
     2281 ggccaccggt gagatcatac cggaagaacg tgatggtttc ctgccaaaaa tcaagcggtg
     2341 gcccggaggc ggcggcgttc gtgaaggtat caatggacgg agcaccgtac ttgaggaaaa
     2401 tcgatttgag gatgtataaa gctagcatgg tgagcaaggg cgaggagctg ttcaccgggg
     2461 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtccgcg
     2521 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg
     2581 gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc gtggcctgct
     2641 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2701 gctacgtcca ggagcgcacc atctctttca aggacgacgg tacctacaag acccgcgccg
     2761 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2821 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaacgtct
     2881 atatcacggc cgacaagcag aagaacggca tcaaggctaa cttcaagatc cgccacaacg
     2941 ttgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3001 gccccgtgct gctgcccgac aaccactacc tgagccatca gtccaagctg agcaaagacc
     3061 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggattacac
     3121 atggcatgga cgagctgtac aagggcgctg gtgcaggcgc cggatccact agtatgaagc
     3181 tacactgtga ggtggaggtg atcagccggc acttgcccgc cttggggctt aggaaccggg
     3241 gcaagggcgt ccgagccgtg ttgagcctct gtcagcagac ttccaggagt cagccgccgg
     3301 tccgagcctt cctgctcatc tccaccctga aggacaagcg cgggacccgc tatgaggtgc
     3361 gtgaagtggg caggccctgt cagtctcgcg ttcttcttgg aagccgagac gcgggccacc
     3421 ctcggtcctc atgctcccgg ctgctcccta ggcgaaagcc cgccttgggg gttcctgaac
     3481 tcccagcctt gagacctacc atcagcccga ccccagggtc ctgtgcgtct tcctacggac
     3541 ccgaaagaag aaagctttga gagtgtacct tttcgctatt tttcctcccc acttttacga
     3601 ctttgaattt acagtgttgc tatttagtag tggatggcaa tcccgcctgt ttcaagtttc
     3661 tgaaattttg cgtgaaacaa gcgcaaatga agcagctgtc cagttgggga acagtaaaat
     3721 aactgggtac ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     3781 tggtcatagc tgtttcctgt gtgaaatttt tatccgctca caattccaca caacatacga
     3841 gccgggagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     3901 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3961 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4021 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4081 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4141 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4201 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4261 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4321 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4381 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4441 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4501 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4561 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4621 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4681 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4741 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4801 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4861 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4921 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4981 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5041 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5101 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5161 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5221 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5281 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5341 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5401 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5461 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5521 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5581 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5641 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5701 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5761 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5821 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5881 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc ac
//
