LOCUS       sequence-459310-        6899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of LZ-SPO11.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6899)
  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 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: "bioRxiv 
            [Preprint]. 2024 Nov 21:2024.11.20.624382. doi: 
            10.1101/2024.11.20.624382."
COMMENT     Sequence Label: pZZMP154
FEATURES             Location/Qualifiers
     source          1..6899
                     /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             961..1017
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1075..1095
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2321..2338)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2327..2551
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2597..3025
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2684..2703)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2894..2915
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3034..3054)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3039..3368
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3219..3354
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3281..3300
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3417..4442
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3417..4442
                     /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    4572..4693
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4609..4628)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4663..4682
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4742..4758)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4742..4758)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4755..4777)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4766..4782
                     /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(4790..4796,4797..4814,4815..4820))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4835..4856
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4973..4990)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5144..5732)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5224..5243)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5903..6694,6695..6763))
                     /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     6526..6545
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6764..6868)
                     /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 aagaggatga aacaactcga agataaagta
      961 gaggagctgc tttccaaaaa ttatcatttg gaaaacgagg tggcacggtt gaagaaattg
     1021 gtaggtgaga ggggttccgg tagcggaggg ggtagcggtg gctctggggg atctgagaac
     1081 ctctacttcc agggagcttt tgcccccatg ggacctgagg ccagcttctt cgatgccctg
     1141 gatagacata gagcctctct gctggccatg gtgaaaagag gcgctggcga gacccctgct
     1201 ggcgccacaa gagtcgcctc cagctccgag gtgctgacag ccatcgaaaa catcatccag
     1261 gacatcatca agtccctggc tagaaacgag gtgcctgcct tcaccatcga caacagatct
     1321 agctgggaga acatcatgtt cgacgatagt gtgggcctgc ggatgatccc ccagtgcacc
     1381 accagaaaga tcagaagcga cagccctaag agcgtgaaga aattcgccct gattctgaag
     1441 gtgctgagca tgatctacaa gctgatccag agcgacacct acgccaccaa gcgggacatc
     1501 tactacaccg actcccagct gttcggcaac caggccgccg tggattctgc catcgatgac
     1561 atcagctgca tgctgaaagt gcccagacgg agcctgcacg tgttgtctac aagcaagggc
     1621 ctgatcgccg gcaatctgcg gtacatggaa gaggacggca cccgggtgca gtgtacctgc
     1681 agcgccaccg ccacagccgt gcctacgaac atccagggca tgcagcacct gatcaccgac
     1741 gccaagttcc tgctgatcgt ggaaaaagat gctaccttcc agcggctgct ggacgacaac
     1801 ttctgcagca gaatgagccc ttgtatcatg gtcacaggca agggcgtgcc agacctgaac
     1861 acccggctgc tggtgaagaa gctgtgggac acatttcaca tcccggtttt caccctggtg
     1921 gacgccgatc cttacggaat cgagatcatg tgcatctata agtacggcag catgagcatg
     1981 agctttgagg cccacaatct gacaattcca acaattcggt ggctgggact gctgcccagc
     2041 gatatccaaa gacttaatat ccccaaggac agcctgatcc ctctgaccaa gcacgaccag
     2101 atgaaactgg acagcatcct gaagagacct tatatcacct accagcctct ctggaagaag
     2161 gaactggaaa tgatggccga ctctaagatg aaggccgaga tccaagctct gacactactg
     2221 agctctgatt acctgtctag agtgtacctg cctaacaagc tgaggttcgg cggatggatc
     2281 taactcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     2341 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     2401 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     2461 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     2521 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     2581 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2641 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2701 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2761 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2821 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2881 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     2941 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     3001 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     3061 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3121 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3181 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3241 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3301 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3361 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcag cacgtgatga
     3421 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
     3481 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
     3541 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt
     3601 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg
     3661 aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag
     3721 acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga
     3781 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg
     3841 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact
     3901 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga
     3961 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca
     4021 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt
     4081 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta
     4141 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc
     4201 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca
     4261 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg
     4321 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg
     4381 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     4441 agcacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4501 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4561 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4621 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4681 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4741 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4801 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4861 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4921 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4981 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5041 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5101 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5161 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5221 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5281 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5341 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5401 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5461 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5521 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5581 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5641 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5701 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5761 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5821 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5881 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5941 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6001 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6061 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6121 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6181 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6241 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6301 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6361 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6421 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6481 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6541 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6601 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6661 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6721 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6781 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6841 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
