LOCUS       sequence-356955-        7307 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of GFP-LacI fusion proteins for LacI/LacO tethering.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7307)
  AUTHORS   Haas J, Bloesel D, Bacher S, Kracht M, Schmitz ML
  TITLE     Chromatin Targeting of HIPK2 Leads to Acetylation-Dependent 
            Chromatin Decondensation.
  JOURNAL   Front Cell Dev Biol. 2020 Sep 1;8:852. doi: 
            10.3389/fcell.2020.00852. eCollection 2020.
  PUBMED    32984337
REFERENCE   2  (bases 1 to 7307)
  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: "Front Cell 
            Dev Biol."; date: "2020-09-1"; volume: "8:852. doi"; pages: " 
            10.3389/fcell.2020.00852. eCollection 2020"
COMMENT     Sequence Label: GFP-LacI-polylinker
FEATURES             Location/Qualifiers
     source          1..7307
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(140..159)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        331..710
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        711..914
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     865..885
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     959..978
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        959..977
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1027..1036
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1033..1035,1036..1038,1039..1749)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1033..1035,1036..1038,1039..1749)
                     /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(1078..1099)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1339..1358)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1686..1707
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1787..1806)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2866..2886
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2994..3011)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3000..3224
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3270..3698
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3357..3376)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3567..3588
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3707..3727)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3712..4041
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3892..4027
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3954..3973
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4108..4902
                     /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(4162..4181)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4772..4791
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5076..5197
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5113..5132)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5167..5186
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5246..5262)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5246..5262)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5259..5281)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5270..5286
                     /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(5294..5300,5301..5318,5319..5324))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5339..5360
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5477..5494)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5648..6236)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5728..5747)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6407..7198,7199..7267))
                     /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     7030..7049
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7268..7307,1..65))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
       61 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
      121 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
      181 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
      241 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
      301 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
      361 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      421 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      481 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      541 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      601 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      661 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      721 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      781 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      841 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      901 atataagcag agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta
      961 atacgactca ctatagggag acccaagctg gctagcgcta ccggggccgc tctagcgcta
     1021 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1081 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1141 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1201 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     1261 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1321 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1381 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1441 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1501 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1561 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1621 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1681 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1741 ctgtacaagt ccggactcag atccatggtg aaaccagtaa cgttatacga tgtcgcagag
     1801 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
     1861 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
     1921 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
     1981 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
     2041 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
     2101 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
     2161 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
     2221 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
     2281 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
     2341 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
     2401 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
     2461 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     2521 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     2581 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     2641 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     2701 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     2761 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     2821 caggtttccc gaagcagcct gagggggcgt cgactgggta cccctcctaa gaagaagagg
     2881 aaggttgccc gggatctagg atccactgtg tctagactta agcttgcggc cgcagaattc
     2941 tcgaggatcg attaagtagc taactagagg gcccgtttaa acccgctgat cagcctcgac
     3001 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3061 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3121 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3181 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3241 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3301 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3361 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3421 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3481 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3541 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3601 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3661 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3721 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3781 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3841 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3901 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3961 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     4021 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     4081 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     4141 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4201 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4261 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4321 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4381 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4441 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4501 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4561 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4621 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4681 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4741 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4801 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4861 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     4921 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     4981 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5041 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5101 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5161 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5221 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5281 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5341 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5401 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5461 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5521 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5581 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5641 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5701 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5761 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5821 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5881 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5941 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6001 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6061 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6121 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6181 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6241 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6301 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6361 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6421 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6481 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6541 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6601 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6661 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6721 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6781 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6841 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6901 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6961 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7021 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7081 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7141 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7201 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7261 tactcatact cttccttttt caatattatt gaagcattta tcagggt
//
