LOCUS       sequence-62970-e        7597 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7597)
  AUTHORS   Liu D, Vleugel M, Backer CB, Hori T, Fukagawa T, Cheeseman IM, 
            Lampson MA
  TITLE     Regulated targeting of protein phosphatase 1 to the outer 
            kinetochore by KNL1 opposes Aurora B kinase.
  JOURNAL   J Cell Biol. 2010 Mar 22;188(6):809-20. doi: 10.1083/jcb.201001006. 
            Epub 2010 Mar 15.
  PUBMED    20231380
REFERENCE   2  (bases 1 to 7597)
  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"; doi: 
            "10.1083/jcb.201001006"; journalName: "J Cell Biol"; date: 
            "2010-03-22- 22"; volume: "188"; issue: "6"; pages: "809-20"
COMMENT     Sequence Label: CENP-B mCherry PP1 gamma
FEATURES             Location/Qualifiers
     source          1..7597
                     /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      903..912
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1422..1431
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1428..2135
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1573..1591)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1841..1860
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(3153..3171)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3154..3171)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3191..3208)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3197..3421
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3467..3895
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3554..3573)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3764..3785
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3904..3924)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3909..4238
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4089..4224
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4151..4170
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4305..5099
                     /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(4359..4378)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4969..4988
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5273..5394
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5310..5329)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5364..5383
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5443..5459)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5443..5459)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5456..5478)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5467..5483
                     /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(5491..5497,5498..5515,5516..5521))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5536..5557
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5674..5691)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5845..6430)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5925..5944)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6601..7392,7393..7461))
                     /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     7224..7243
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7462..7566)
                     /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 gcttggcgcg
      901 ccgccaccat gggccccaag aggcgacagc tgacgttccg ggagaagtca cggatcatcc
      961 aggaggtgga ggagaatccg gacctgcgca agggcgagat cgcgcggcgc ttcaacatcc
     1021 cgccgtccac gctgagcacg atcctgaaga acaagcgcgc catcctggcg tcggagcgca
     1081 agtacggggt ggcctccacc tgccgcaaga ccaacaagct gtctccctac gacaagctcg
     1141 agggcttgct catcgcctgg ttccagcaga tccgcgccgc cggcctgccg gtcaagggca
     1201 tcatcctcaa ggagaaggcg ctgcgcatag ccgaggagct gggcatggac gacttcaccg
     1261 cctccaacgg ctggctggac cgcttccgcc ggcgccacgg cgtggtgtcc tgcagcggcg
     1321 tggcccgcgc ccgcgcgcga aacgctgccc cccgcacccc ggcggcgcct gccagtccgg
     1381 ccgcggtgcc ctcggagggc agtggcgggc tagcatgctt cgccaccatg gtgagcaagg
     1441 gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
     1501 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     1561 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     1621 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     1681 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     1741 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     1801 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     1861 ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     1921 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     1981 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     2041 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     2101 gccactccac cggcggcatg gacgagctgt acaagagatc tcgagctcaa gcttcgaatt
     2161 cgatggcgga tttagataaa ctcaacatcg acagcattat ccaacggctg ctggaagtga
     2221 gagggtccaa gcctggtaag aatgtccagc ttcaggagaa tgaaatcaga ggactgtgct
     2281 taaagtctcg tgaaatcttt ctcagtcagc ctatcctact agaacttgaa gcaccactca
     2341 aaatatgtgg tgacatccat ggacaatact atgatttgct gcgacttttt gagtacggtg
     2401 gtttcccacc agaaagcaac tacctgtttc ttggggacta tgtggacagg ggaaagcagt
     2461 cattggagac gatctgcctc ttactggcct acaaaataaa atatcctgag aatttttttc
     2521 ttctcagagg gaaccatgaa tgtgccagca tcaacagaat ttatggattt tatgatgaat
     2581 gtaaaagaag atacaacatt aaactatgga aaactttcac agactgtttt aactgtttac
     2641 cgatagcagc catcgtggat gagaagatat tctgttgtca tggaggttta tcaccagatc
     2701 ttcaatctat ggagcagatt cggcgaatta tgcgaccaac tgatgtacca gatcaaggtc
     2761 ttctttgtga tcttttgtgg tctgaccccg ataaagatgt cttaggctgg ggtgaaaatg
     2821 acagaggagt gtccttcaca tttggtgcag aagtggttgc aaaatttctc cataagcatg
     2881 atttggatct tatatgtaga gcccatcagg tggttgaaga tggatatgaa ttttttgcaa
     2941 agaggcagtt ggtcactctg ttttctgcgc ccaattattg cggagagttt gacaatgcag
     3001 gtgccatgat gagtgtggat gaaacactaa tgtgttcttt tcagatttta aagcctgcag
     3061 agaaaaagaa gccaaatgcc acgagacctg taacgcctcc aaggggtatg atcacaaagc
     3121 aagcaaagaa ataggaatct agagggccct attctatagt gtcacctaaa tgctagagct
     3181 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3241 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3301 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3361 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3421 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3481 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3541 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3601 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3661 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3721 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3781 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3841 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     3901 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3961 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     4021 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     4081 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     4141 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     4201 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     4261 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     4321 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4381 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4441 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     4501 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4561 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4621 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4681 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4741 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4801 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     4861 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4921 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4981 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     5041 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     5101 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     5161 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     5221 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     5281 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5341 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     5401 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5461 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5521 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5581 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5641 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5701 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5761 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5821 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5881 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5941 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6001 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6061 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6121 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6181 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6241 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6301 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6361 acaaaccacc gctggtagcg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6421 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6481 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6541 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6601 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6661 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6721 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6781 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6841 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6901 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6961 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     7021 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     7081 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7141 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7201 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7261 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7321 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7381 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7441 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7501 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7561 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
