LOCUS       sequence-365827-        7582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of recombinant beta1 integrin cytosolic domain with 
            C-terminal GFP tag in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7582)
  AUTHORS   Case LB, De Pasquale M, Henry L, Rosen MK
  TITLE     Synergistic phase separation of two pathways promotes integrin 
            clustering and nascent adhesion formation.
  JOURNAL   Elife. 2022 Jan 20;11. pii: 72588. doi: 10.7554/eLife.72588.
  PUBMED    35049497
REFERENCE   2  (bases 1 to 7582)
  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: "Elife."; 
            date: "2022-01-20"; volume: "11. pii: 72588. doi"; pages: " 
            10.7554/eLife.72588"
COMMENT     Sequence Label: B1 Integrin-GFP
FEATURES             Location/Qualifiers
     source          1..7582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     530..549
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(569..646)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     852..871
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     955..974
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        955..973
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    974..998
                     /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)."
     RBS             1013..1035
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1051..1080
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1084..2184
                     /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="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2041..2050
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2158..2181
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2233..2244
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2251..2271
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2476..2478,2479..2481,2482..3195)
                     /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(2521..2542)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2782..2801)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3129..3150
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3205..3225
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3268..3285
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3338..3356)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3352..3399
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3436..3891
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3523..3542)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3733..3754
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3917..4021
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4022..4090,4091..4882)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4240..4259)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5053..5641
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5542..5561
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5795..5812
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5827..5966
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5892..5911)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6052..6074
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6068..6259)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7068..7582,1..568))
                     /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"
ORIGIN
        1 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
       61 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
      121 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
      181 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
      241 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
      301 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
      361 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
      421 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
      481 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
      541 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
      601 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
      661 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
      721 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
      781 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
      841 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
      901 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
      961 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     1021 actttaagaa ggagatatac catgtctggc catcaccatc accatcacca tcaccatcac
     1081 ggcatgaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac
     1141 ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag
     1201 catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac
     1261 attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa
     1321 atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt
     1381 tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac
     1441 aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa
     1501 ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg
     1561 ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt
     1621 aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg
     1681 attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat
     1741 aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc
     1801 aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc
     1861 gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag
     1921 ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg
     1981 ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc
     2041 gccaccatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct
     2101 ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat
     2161 gaagccctga aagacgcgca gactaattcg agctcgaaca acaacaacaa taacaataac
     2221 aacaacctcg ggatcgaggg aaggatttca gaaaacctgt acttccaggg atccggtgga
     2281 ggcggatgta aactgcttat gatcattcat gatcgccgcg aatttgccaa atttgagaag
     2341 gaaaaaatga acgctaaatg ggatacgggc gaaaacccca tttacaagtc agctgtgacg
     2401 accgttgtaa acccgaagta tgaggggaaa ggtgggagta gtggtggtag cggtggcagt
     2461 ggaggtagcg aattcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     2521 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     2581 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     2641 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     2701 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     2761 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     2821 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     2881 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     2941 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     3001 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     3061 ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc caacgagaag
     3121 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     3181 gagctgtaca agtagtgata actcgagaac ctgtattttc agggccgtaa acgtaaacgt
     3241 aaacgtaaac gtaaaggctg agtcgagcac caccaccacc accactgaga tccggctgct
     3301 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     3361 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     3421 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3481 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3541 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3601 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3661 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3721 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3781 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3841 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     3901 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3961 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     4021 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     4081 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4141 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4201 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4261 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4321 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4381 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4441 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4501 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4561 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4621 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4681 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4741 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4801 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4861 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4921 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4981 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5041 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5101 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5161 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     5221 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5281 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5341 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5401 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5461 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5521 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5581 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5641 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5701 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5761 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5821 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     5881 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     5941 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     6001 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     6061 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     6121 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     6181 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     6241 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     6301 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     6361 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     6421 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     6481 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     6541 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     6601 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     6661 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     6721 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     6781 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     6841 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     6901 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     6961 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     7021 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     7081 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     7141 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc
     7201 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     7261 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct
     7321 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     7381 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     7441 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     7501 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     7561 cgcagacgcg ccgagacaga ac
//
