LOCUS       sequence-224869-        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"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     complement(255..274)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             1316..1336
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1355..1364
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1361..1363,1364..1366,1367..2077)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1361..1363,1364..1366,1367..2077)
                     /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(1406..1427)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1667..1686)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2014..2035
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2123..2143
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2156..2200
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     misc_feature    2219..2271
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2395..2516
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2432..2451)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2486..2505
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2523..2978)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2660..2681)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2872..2891
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3005..3109
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3111..3468
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3111..3302
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3134..3154)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3319..3454
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3503..4297
                     /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             3503..4297
                     /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(3557..3576)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4167..4186
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4485..4504)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4529..4576
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4905..5493
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5394..5413
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        5668..5971
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcatggtgaa
      241 accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg
      301 cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat
      361 ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt
      421 gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc
      481 gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag
      541 cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct
      601 gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa
      661 tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc
      721 ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat
      781 cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca
      841 taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc
      901 catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat
      961 gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct
     1021 gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta
     1081 tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga
     1141 ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc
     1201 actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt
     1261 ggccgattca ttaatgcagc tggcacgaca ggtttcccga agcagcctga ggcctcctaa
     1321 gaagaagagg aaggttgggc tagcgctacc ggtcgccacc atggtgagca agggcgagga
     1381 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     1441 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     1501 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     1561 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     1621 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     1681 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1741 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1801 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     1861 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     1921 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     1981 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     2041 cgccgggatc actctcggca tggacgagct gtacaagtcc ggaggggatc ggtggagctc
     2101 caccggtggc ggccgctcta gagagaatct ttattttcag ggcgccgcca aattcaaaga
     2161 aaccgctgct gctaaattcg aacgccagca catggacagc ggaggtggag gtactagtct
     2221 cgagctcaag cttcgaattc tgcagtcgac ggtaccgcgg gcccgggatc caccggatct
     2281 agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     2341 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     2401 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     2461 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc
     2521 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     2581 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     2641 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     2701 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     2761 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     2821 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     2881 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     2941 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     3001 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3061 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     3121 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3181 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3241 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3301 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3361 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     3421 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     3481 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     3541 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     3601 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     3661 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     3721 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     3781 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     3841 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     3901 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     3961 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     4021 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     4081 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     4141 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     4201 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     4261 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     4321 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4381 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4441 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     4501 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     4561 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     4621 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     4681 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     4741 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4801 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4861 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4921 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4981 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5041 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5101 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5161 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5221 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5281 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5341 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5401 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5461 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5521 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     5581 gtggataacc gtattaccgc catgcattag ttattaatag taatcaatta cggggtcatt
     5641 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     5701 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     5761 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     5821 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     5881 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     5941 catctacgta ttagtcatcg ctattaccat g
//
