LOCUS       sequence-459305-        7277 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of TOP6BL.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7277)
  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 7277)
  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: pZZMP26
FEATURES             Location/Qualifiers
     source          1..7277
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             136..1161
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             136..1161
                     /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    1291..1412
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1328..1347)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1382..1401
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1461..1477)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1461..1477)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1474..1496)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1485..1501
                     /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(1509..1515,1516..1533,1534..1539))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1554..1575
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1692..1709)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1863..2451)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1943..1962)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2622..3413,3414..3482))
                     /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     3245..3264
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3483..3587)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3662..3681)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3853..4232
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4233..4436
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4387..4407
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4481..4500
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4481..4499
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4519..4528
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(6317..6334)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6323..6547
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6593..7021
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6680..6699)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6890..6911
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7030..7050)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(7035..7277,1..87)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(7215..7277,1..73)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     join(7277,1..19)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
       61 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
      121 tctgatcagc acgtgatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
      181 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
      241 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
      301 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
      361 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
      421 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
      481 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
      541 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
      601 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
      661 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
      721 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
      781 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
      841 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
      901 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
      961 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     1021 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     1081 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     1141 cgtccgaggg caaaggaata gcacgtgcta cgagatttcg attccaccgc cgccttctat
     1201 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     1261 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     1321 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     1381 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     1441 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     1501 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     1561 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1621 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1681 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     1741 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     1801 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1861 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1921 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1981 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2041 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2101 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2161 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2221 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2281 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2341 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2401 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2461 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2521 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2581 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2641 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2701 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2761 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2821 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2881 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2941 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3001 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3061 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3121 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3181 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3241 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3301 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3361 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3421 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3481 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3541 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     3601 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     3661 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     3721 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     3781 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     3841 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     3901 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     3961 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     4021 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     4081 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     4141 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     4201 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     4261 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     4321 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     4381 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct
     4441 ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact cactataggg
     4501 agacccaagc tggctagcgc caccatgggc gaacggaccg ccctggccgt gtgcgaaatc
     4561 ctgagatacc tgatcatcca ctggaagtgc gaagccggca ccgccaaggg caccctgctg
     4621 gacggccagc tggtgatctc tatcgaggcc ctgagaagca agcacctgcc cgacagcctg
     4681 cattgcatca ttaccatcgc cagcacccgc agcgtctacg gcgggctgaa cttcaagaag
     4741 ttcctgcagg agatccaacc tgccctgcct agactgagcg ctaagctggc cctggcctct
     4801 gaggaaggcg gcagaagcca agatgcctct ggcatcgccc cgtgccaggt gaccttcgag
     4861 gtggacgaga actctcaatc cctgatgacc gactgcctgg tcatcaagca cttcctgcgg
     4921 aagatcatca tcgtgcacca caagctgaaa ttctccttca gcgtggccgt gaatggcaca
     4981 ctgtccgccg agacattcgg cgccgaaaac gagcctacac tgcgcctgga taacggcgtg
     5041 acactggtgg tgggctttca gcggtacgtg agcaagccta agctgaactg gagcgaggct
     5101 cactgcagca gaatccaccc cgtgctggga caccctgccc ctctgtttat ccctgacgcc
     5161 aaagccgata ccggcctgct gggcgagctg acactgaccc ctgctgctgc cctctgtcct
     5221 agcccaaaag ggttttcttc tcagctgtgt agaatcagca gcgtgtctat tttcctctat
     5281 ggccccctgg gcctgccact gcttagctcc gatcaggacc agcccagcac agccgtgttc
     5341 agagatacaa gctacttcat cgactggaaa aaatacaacc tgttcatggt gcctaacctg
     5401 gacctgaatc tggacactca gagcgtcctg cccgacgtga attacaaggc cgaaagcccc
     5461 gagggcaacc agtcccagaa tatgaacgcc cagggacctg ctctgctgct gtttctgttc
     5521 gtggacttcc agagcgacgt gcccgttcag caagctaaaa tctggggcct ccacaccctg
     5581 ctgaccgccc acctgagcgc catcctgagc gagagccggt ctacagtgca gcagtctatc
     5641 cagagcgccg tggatcaggt gtggcagctg taccaccatg atgccaaaac ccagcagaga
     5701 ctgcaggcta gcctgagcgt ggccgtgaac agcatcatga gcgtgctgac cggctctacc
     5761 agaagctctt ttagaaagac ctgcttgcag gcactggaag ccgccgacac ccaggaattc
     5821 ggcgttaagc tgcaccggat cttctacgac atcacacagc accagttcct caagcactgt
     5881 agctgcgaca cagagcagca cctgaccccc gagaagaaca tcagcgccca gaacacaaag
     5941 gaccagcata agaacatcgc ccaggagttc cctgaagaaa gcataggaca ggccgagaac
     6001 aagagaccta agcggggcag ccctaaccac ggacgggaag agtcaagagt gctgggcagc
     6061 gcacgggaca gaagtcctcc taagagcgcc accagggata gagaactgac cgaggtgtcc
     6121 ctgacggcca gaggaagcca gacccaggcc gctcacggcc gggcccaagc cgccgaggcc
     6181 gcttctccag ctggcggact ggaagatctg tggctgcaag aggtgtccaa cctgtctgag
     6241 tggctgaacc ctggccacag aagttaagga tccatcgatc tcgagtctag agggcccgtt
     6301 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     6361 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     6421 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     6481 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     6541 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     6601 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     6661 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     6721 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     6781 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     6841 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     6901 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     6961 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     7021 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     7081 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     7141 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     7201 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     7261 gctgactaat ttttttt
//
