LOCUS       sequence-10521-e        7435 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 7435)
  AUTHORS   Stumpff J, von Dassow G, Wagenbach M, Asbury C, Wordeman L
  TITLE     The kinesin-8 motor Kif18A suppresses kinetochore movements to 
            control mitotic chromosome alignment.
  JOURNAL   Dev Cell. 2008 Feb . 14(2):252-62.
  PUBMED    18267093
REFERENCE   2  (bases 1 to 7435)
  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: "Dev Cell. 
            2008 Feb . 14(2):252-62."
COMMENT     Sequence Label: pMX229
FEATURES             Location/Qualifiers
     source          1..7435
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4063..4080
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    4223..4344
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4260..4279)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4314..4333
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4351..4806)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4488..4509)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4700..4719
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4833..4937
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4939..5296
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4939..5130
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4962..4982)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5147..5282
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5331..6125
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5331..6125
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5385..5404)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5995..6014
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6313..6332)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6357..6404
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6733..7321
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7222..7241
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct caagcttcga attccaccat gtctgtcact
     1381 gaggaagacc tgtgccacca tatgaaagta gtagttcgtg tacgtccgga aaacactaaa
     1441 gaaaaagcag ctggatttca taaagtggtt catgttgtgg ataaacatat cctagttttt
     1501 gatcccaaac aagaagaagt cagttttttc catggaaaga aaactacaaa tcaaaatgtt
     1561 ataaagaaac aaaataagga tcttaaattt gtatttgatg ctgtttttga tgaaacgtca
     1621 actcagtcag aagtttttga acacactact aagccaattc ttcgtagttt tttgaatgga
     1681 tataattgca cagtacttgc ctatggtgcc actggtgctg ggaagaccca cactatgcta
     1741 ggatcagctg atgaacctgg agtgatgtat ctaacaatgt tacaccttta caaatgcatg
     1801 gatgagatta aagaagagaa aatatgtagt actgcagttt catatctgga ggtatataat
     1861 gaacagattc gtgatctctt agtaaattca gggccacttg ctgtccggga agatacccaa
     1921 aaaggggtgg tcgttcatgg acttacttta caccagccca aatcctcaga agaaatttta
     1981 catttattgg ataatggaaa caaaaacagg acacaacatc ccactgatat gaatgccaca
     2041 tcttctcgtt ctcatgctgt tttccaaatt tacttgcgac aacaagacaa aacagcaagt
     2101 atcaatcaaa atgtccgtat tgccaagatg tcactcattg acctggcagg atctgagcga
     2161 gcaagtactt ccggtgctaa ggggacccga tttgtagaag gcacaaatat taatagatca
     2221 cttttagctc ttgggaatgt catcaatgcc ttagcagatt caaagagaaa gaatcagcat
     2281 atcccttaca gaaatagtaa gcttactcgc ttgttaaagg attctcttgg aggaaactgt
     2341 caaactataa tgatagctgc tgttagtcct tcctctgtat tctacgatga cacatataac
     2401 actcttaagt atgctaaccg ggcaaaggac attaaatctt ctttgaagag caatgttctt
     2461 aatgtcaata atcatataac tcaatatgta aagatctgta atgagcagaa ggcagagatt
     2521 ttattgttaa aagaaaaact aaaagcctat gaagaacaga aagccttcac taatgaaaat
     2581 gaccaagcaa agttaatgat ttcaaaccct caggaaaaag aaatcgaaag gtttcaagaa
     2641 atcctgaact gcttgttcca gaatcgagaa gaaattagac aagaatatct gaagttggaa
     2701 atgttactta aagaaaatga acttaaatca ttctaccaac aacagtgcca taaacaaata
     2761 gaaatgatgt gttctgaaga caaagtagaa aaggccactg gaaaacgaga tcatagactt
     2821 gcaatgttga aaactcgtcg ctcctacctg gagaaaagga gggaggagga attgaagcaa
     2881 tttgatgaga atactaattg gctccatcgt gtcgaaaaag aaatgggact cttaagtcaa
     2941 aacggtcata ttccaaagga actcaagaaa gatcttcatt gtcaccattt gcacctccag
     3001 aacaaagatt tgaaagcaca aattagacat atgatggatc tagcttgtct tcaggaacag
     3061 caacacaggc agactgaagc agtattgaat gctttacttc caaccctaag aaaacaatat
     3121 tgcacattaa aagaagccgg cctgtcaaat gctgcttttg aatctgactt caaagagatc
     3181 gaacatttgg tagagaggaa aaaagtggta gtttgggctg accaaactgc cgaacaacca
     3241 aagcaaaacg atctaccagg gatttctgtt cttatgacct ttccacaact tggaccagtt
     3301 cagcctattc cttgttgctc atcttcaggt ggaactaatc tggttaagat tcctacagaa
     3361 aaaagaactc ggagaaaact aatgccatct cccttgaaag gacagcatac tctaaagtct
     3421 ccaccatctc aaagtgtgca gctcaatgat tctcttagca aagaacttca gcctattgta
     3481 tatacaccag aagactgtag aaaagctttt caaaatccgt ctacagtaac cttaatgaaa
     3541 ccatcatcat ttactacaag ttttcaggct gtcagctcaa acataaacag tgataattgt
     3601 ctgaaaatgt tgtgtgaagt agctatccct cataatagaa gaaaagaatg tggacaggag
     3661 gacttggact ctacatttac tatatgtgaa gacatcaaga gctcgaagtg taaattaccc
     3721 gaacaagaat cactaccaaa tgataacaaa gacattttac aacggcttga tccttcttca
     3781 ttctcaacta agcattctat gcctgtacca agcatggtgc catcctacat ggcaatgact
     3841 actgctgcca aaaggaaacg gaaattaaca agttctacat caaacagttc gttaactgca
     3901 gacgtaaatt ctggatttgc caaacgtgtt cgacaagata attcaagtga gaagcactta
     3961 caagaaaaca aaccaacaat ggaacataaa agaaacatct gtaaaataaa tccaagcatg
     4021 gttagaaaat ttggaagaaa tatttcaaaa ggaaatctaa gacatcacca ccatcaccat
     4081 taagcggccg cggggatcca ccggatctag ataactgatc ataatcagcc ataccacatt
     4141 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     4201 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     4261 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4321 gtccaaactc atcaatgtat cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat
     4381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     4441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     4501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     4561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     4621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     4681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     4741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     4801 gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4861 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4921 attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg
     4981 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     5041 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     5101 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     5161 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     5221 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     5281 cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac
     5341 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     5401 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     5461 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg
     5521 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     5581 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     5641 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     5701 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     5761 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     5821 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc
     5881 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     5941 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     6001 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     6061 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     6121 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     6181 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     6241 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag
     6301 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc
     6361 aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg
     6421 gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg
     6481 cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc
     6541 caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga
     6601 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6661 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6721 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6781 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6841 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     6901 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6961 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7021 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7081 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7141 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7201 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7261 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7321 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7381 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcat
//
