LOCUS       sequence-307849-        7551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mEGFP and LANA peptide fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7551)
  AUTHORS   Gibson BA, Doolittle LK, Schneider MWG, Jensen LE, Gamarra N, Henry 
            L, Gerlich DW, Redding S, Rosen MK
  TITLE     Organization of Chromatin by Intrinsic and Regulated Phase 
            Separation.
  JOURNAL   Cell. 2019 Oct 3;179(2):470-484.e21. doi: 
            10.1016/j.cell.2019.08.037. Epub 2019 Sep 19.
  PUBMED    31543265
REFERENCE   2  (bases 1 to 7551)
  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: 
            "10.1016/j.cell.2019.08.037"; journalName: "Cell"; date: "2019-10-3-
            3"; volume: "179"; issue: "2"; pages: "470-484.e21"
COMMENT     Sequence Label: pMTTH+mEGFP-LANA
FEATURES             Location/Qualifiers
     source          1..7551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(89..94,95..110,111..117)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    125..141
                     /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)."
     primer_bind     130..152
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             211..1311
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1168..1177
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1285..1308
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1360..1371
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1384..1404
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1408..1410,1411..1413,1414..2124)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1453..1474)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1714..1733)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2061..2082
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2251..2271
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2272..2289
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2302..2319)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2302..2318)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2311..2333)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2497..2514)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2667..2753
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2845..2872
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2891..2982
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2983..3051,3052..3843)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3201..3220)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3885..4398)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4080..4101)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4292..4311
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4509..5097
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4998..5017
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5251..5268
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5374..5393)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5534..5556
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5550..5741)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6237..6314
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             6315..7397
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(6334..6353)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tcgtgtcgct caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg
       61 gttctggcaa atattctgaa atgagctgtt gacaattaat catcggctcg tataatgtgt
      121 ggaattgtga gcggataaca atttcacaca ggaaacagcc agtccgttta ggtgttttca
      181 cgagcacttc accaacaagg accatagatt atgaaaactg aagaaggtaa actggtaatc
      241 tggattaacg gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa
      301 gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag
      361 gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc
      421 tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg
      481 tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct
      541 gttgaagcgt tatcgctgat ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg
      601 gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc
      661 aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc
      721 aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa
      781 gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat
      841 tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg
      901 tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc
      961 ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc
     1021 agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac tgatgaaggt
     1081 ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa
     1141 gagttggcga aagatccacg tattgccgcc accatggaaa acgcccagaa aggtgaaatc
     1201 atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac
     1261 gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac taattcgagc
     1321 tcgaacaaca acaacaataa caataacaac aacctcggga tcgagggaag gatttcagaa
     1381 tttgaaaacc tgtattttca gggccatatg gtgagcaagg gcgaggagct gttcaccggg
     1441 gtggtgccta tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1501 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1561 ggcaagctgc cagtgccgtg gcctaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     1621 ttcagccgct acccagacca catgaagcag cacgacttct tcaagtccgc catgccggaa
     1681 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     1741 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1801 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1861 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1921 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc tatcggcgac
     1981 ggcccagtgc tgctgccgga caaccactac ctgagcaccc agtccaaact gagcaaagac
     2041 cctaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     2101 ctcggcatgg acgagctgta caagggaggc agtgggacgg gaggtagtgg cactggaggt
     2161 tctgggacta tggcgcctcc tggcatgcgt ttgcgttccg gccgtagtac cggggcgcca
     2221 cttacgcgtg gatctggctc gagcggatcc gaaaacctgt attttcaggg ccatcatcat
     2281 catcatcact gaaagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     2341 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     2401 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggca
     2461 gcttggctgt tttggcggat gagataagat tttcagcctg atacagatta aatcagaacg
     2521 cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga
     2581 ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca
     2641 tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     2701 cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg
     2761 gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat
     2821 aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc
     2881 tacaaactct tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2941 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     3001 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     3061 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3121 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg tttcccaatg
     3181 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     3241 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3301 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3361 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     3421 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     3481 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     3541 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     3601 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     3661 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     3721 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     3781 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     3841 taactgtcag accaagttta ctcatatata ctttagattg atttaccccg gttgataatc
     3901 agaaaagccc caaaaacagg aagattgtat aagcaaatat ttaaattgta aacgttaata
     3961 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     4021 aaatcggcaa aatcccttat aaatcaaaag aatagcccga gatagggttg agtgttgttc
     4081 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     4141 ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt
     4201 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     4261 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     4321 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     4381 cgccgctaca gggcgcgtaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4441 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4501 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4561 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4621 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4681 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4741 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4801 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4861 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4921 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4981 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5041 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5101 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5161 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     5221 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     5281 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catgtggtgc
     5341 actctcagta caccgcatgt ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5401 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     5461 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5521 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5581 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgca gcgattcaca gatgtctgcc
     5641 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     5701 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     5761 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     5821 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     5881 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     5941 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     6001 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     6061 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     6121 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     6181 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga aattccgaca
     6241 ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
     6301 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
     6361 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
     6421 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
     6481 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
     6541 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
     6601 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
     6661 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
     6721 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
     6781 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
     6841 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
     6901 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
     6961 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
     7021 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
     7081 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
     7141 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     7201 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     7261 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     7321 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     7381 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcacaa
     7441 ttctcatgtt tgacagctta tcatcgactg cacggtgcac caatgcttct ggcgtcaggc
     7501 agccatcgga agctgtggta tggctgtgca ggtcgtaaat cactgcataa t
//
