LOCUS       sequence-459308-        6770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of SPO11.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6770)
  AUTHORS   Zheng Z, Zheng L, Arter M, Liu K, Yamada S, Ontoso D, Kim S, Keeney 
            S
  TITLE     Reconstitution of SPO11-dependent double-strand break formation.
  JOURNAL   bioRxiv [Preprint]. 2024 Nov 21:2024.11.20.624382. doi: 
            10.1101/2024.11.20.624382.
  PUBMED    39605552
REFERENCE   2  (bases 1 to 6770)
  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: "bioRxiv 
            [Preprint]. 2024 Nov 21:2024.11.20.624382. doi: 
            10.1101/2024.11.20.624382."
COMMENT     Sequence Label: pZZMP09
FEATURES             Location/Qualifiers
     source          1..6770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             910..933
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             934..954
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2192..2209)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2198..2422
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2468..2896
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2555..2574)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2765..2786
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2905..2925)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2910..3239
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3090..3225
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3152..3171
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3288..4313
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3288..4313
                     /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"
     polyA_signal    4443..4564
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4480..4499)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4534..4553
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4613..4629)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4613..4629)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4626..4648)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4637..4653
                     /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(4661..4667,4668..4685,4686..4691))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4706..4727
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4844..4861)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5015..5603)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5095..5114)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5774..6565,6566..6634))
                     /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     6397..6416
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6635..6739)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccaccatgg attacaagga tgacgacgat aaggagaacc tctacttcca gggagctttt
      961 gcccccatgg gacctgaggc cagcttcttc gatgccctgg atagacatag agcctctctg
     1021 ctggccatgg tgaaaagagg cgctggcgag acccctgctg gcgccacaag agtcgcctcc
     1081 agctccgagg tgctgacagc catcgaaaac atcatccagg acatcatcaa gtccctggct
     1141 agaaacgagg tgcctgcctt caccatcgac aacagatcta gctgggagaa catcatgttc
     1201 gacgatagtg tgggcctgcg gatgatcccc cagtgcacca ccagaaagat cagaagcgac
     1261 agccctaaga gcgtgaagaa attcgccctg attctgaagg tgctgagcat gatctacaag
     1321 ctgatccaga gcgacaccta cgccaccaag cgggacatct actacaccga ctcccagctg
     1381 ttcggcaacc aggccgccgt ggattctgcc atcgatgaca tcagctgcat gctgaaagtg
     1441 cccagacgga gcctgcacgt gttgtctaca agcaagggcc tgatcgccgg caatctgcgg
     1501 tacatggaag aggacggcac ccgggtgcag tgtacctgca gcgccaccgc cacagccgtg
     1561 cctacgaaca tccagggcat gcagcacctg atcaccgacg ccaagttcct gctgatcgtg
     1621 gaaaaagatg ctaccttcca gcggctgctg gacgacaact tctgcagcag aatgagccct
     1681 tgtatcatgg tcacaggcaa gggcgtgcca gacctgaaca cccggctgct ggtgaagaag
     1741 ctgtgggaca catttcacat cccggttttc accctggtgg acgccgatcc ttacggaatc
     1801 gagatcatgt gcatctataa gtacggcagc atgagcatga gctttgaggc ccacaatctg
     1861 acaattccaa caattcggtg gctgggactg ctgcccagcg atatccaaag acttaatatc
     1921 cccaaggaca gcctgatccc tctgaccaag cacgaccaga tgaaactgga cagcatcctg
     1981 aagagacctt atatcaccta ccagcctctc tggaagaagg aactggaaat gatggccgac
     2041 tctaagatga aggccgagat ccaagctctg acactactga gctctgatta cctgtctaga
     2101 gtgtacctgc ctaacaagct gaggttcggc ggatggatct aaggatccat cgatctcgag
     2161 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     2221 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     2281 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     2341 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     2401 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     2461 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2521 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2581 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2641 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2701 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2761 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2821 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2881 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     2941 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3001 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3061 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3121 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     3181 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     3241 agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgatg aaaaagcctg
     3301 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
     3361 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
     3421 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc
     3481 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg
     3541 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg
     3601 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg
     3661 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca
     3721 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg
     3781 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
     3841 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc
     3901 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
     3961 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc
     4021 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt
     4081 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
     4141 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg
     4201 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac
     4261 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagcacgtgc
     4321 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4381 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4441 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4501 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4561 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4621 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4681 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4741 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4801 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4861 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4921 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4981 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5041 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5101 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5161 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5221 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5281 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5341 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5401 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5461 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5521 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5581 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5641 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5701 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5761 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5821 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5881 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5941 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6001 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6061 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6121 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6181 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6241 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6301 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6361 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6421 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6481 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6541 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6601 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6661 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6721 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
