LOCUS       sequence-218805-        5971 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for expressing LacI-LAP-fused proteins..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5971)
  AUTHORS   Vleugel M, Tromer E, Omerzu M, Groenewold V, Nijenhuis W, Snel B, 
            Kops GJ
  TITLE     Arrayed BUB recruitment modules in the kinetochore scaffold KNL1 
            promote accurate chromosome segregation.
  JOURNAL   J Cell Biol. 2013 Dec 23;203(6):943-55. doi: 10.1083/jcb.201307016.
  PUBMED    24344183
REFERENCE   2  (bases 1 to 5971)
  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"; 
            journalName: "J Cell Biol"; date: "2013-12-23- 23"; volume: "203"; 
            issue: "6"; pages: "943-55"
COMMENT     Sequence Label: pLacI-LAP
FEATURES             Location/Qualifiers
     source          1..5971
                     /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"
     primer_bind     complement(619..638)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             1680..1700
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1719..1728
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1725..1727,1728..1730,1731..2441)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1725..1727,1728..1730,1731..2441)
                     /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(1770..1791)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2031..2050)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2378..2399
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2487..2507
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2520..2564
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     misc_feature    2583..2635
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2759..2880
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2796..2815)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2850..2869
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2887..3342)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3024..3045)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3236..3255
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3369..3473
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3475..3832
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3475..3666
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3498..3518)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3683..3818
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3867..4661
                     /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             3867..4661
                     /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(3921..3940)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4531..4550
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4849..4868)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4893..4940
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5269..5857
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5758..5777
                     /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 gctagcatgg
      601 tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt
      661 cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg
      721 cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt
      781 cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg
      841 cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac
      901 gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg
      961 ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca
     1021 ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt
     1081 tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc
     1141 aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct
     1201 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
     1261 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
     1321 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
     1381 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
     1441 gttatatccc gccgtcaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
     1501 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
     1561 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
     1621 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgaagcagc ctgaggcctc
     1681 ctaagaagaa gaggaaggtt gggctagcgc taccggtcgc caccatggtg agcaagggcg
     1741 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1801 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     1861 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1921 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1981 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     2041 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     2101 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     2161 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     2221 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     2281 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     2341 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     2401 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtccggaggg gatcggtgga
     2461 gctccaccgg tggcggccgc tctagagaga atctttattt tcagggcgcc gccaaattca
     2521 aagaaaccgc tgctgctaaa ttcgaacgcc agcacatgga cagcggaggt ggaggtacta
     2581 gtctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg gatccaccgg
     2641 atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     2701 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     2761 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     2821 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     2881 acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2941 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     3001 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     3061 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     3121 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     3181 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     3241 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     3301 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     3361 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3421 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg
     3481 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     3541 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3601 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3661 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3721 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     3781 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc
     3841 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3901 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3961 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     4021 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     4081 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     4141 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     4201 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     4261 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4321 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4381 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4441 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4501 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4561 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     4621 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     4681 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     4741 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4801 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     4861 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     4921 cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga
     4981 taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca
     5041 ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc
     5101 catagcctca ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5161 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5221 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5281 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5341 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5401 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5461 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5521 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5581 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5641 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5701 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5761 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5821 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5881 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     5941 ttctgtggat aaccgtatta ccgccatgca t
//
