LOCUS       sequence-365813-        6924 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of recombinant nephrin in bacteria cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6924)
  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 6924)
  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: Nephrin
FEATURES             Location/Qualifiers
     source          1..6924
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(93..179)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     332..349
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     primer_bind     513..535
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     527..544
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     528..544
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(557..574)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(575..595)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(629..652)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(815..835)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(848..859)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(908..2008)
                     /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"
     primer_bind     complement(911..934)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      1042..1051
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2067..2089)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2078..2094
                     /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(2102..2108,2109..2124,2125..2130))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(2373..3455)
                     /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     3417..3436
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3456..3533)
                     /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             4029..4220
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4214..4236)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4377..4396
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4502..4519)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4673..5261)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4753..4772)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     rep_origin      5372..5885
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5459..5478)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5669..5690
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5927..6718,6719..6787))
                     /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     6550..6569
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6788..6879)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(join(6898..6924,1))
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 tgcttaattt gatgcctggc agtttatggc gggcgtcctg cccgccaccc tccgggccgt
       61 tgcttcgcaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag agcgttcacc
      121 gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga
      181 tgcctggcag ttccctactc tcgcatgggg agaccccaca ctaccatcgg cgctacggcg
      241 tttcacttct gagttcggca tggggtcagg tgggaccacc gcgctactgc cgccaggcaa
      301 attctgtttt atcagaccgc ttctgcgttc tgatttaatc tgtatcaggc tgaaaatctt
      361 atctcatccg ccaaaacagc caagctgcca ttcgccattc aggctgcgca actgttggga
      421 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
      481 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
      541 cagtgccaag ctttcagtga tgatgatgat gatggccctg aaaatacagg ttttcggaac
      601 ggcgacggcg acggcgacgg cttccacccg gaccctgaaa cagcacttcc agggatccat
      661 cgcccgccac ctgatcataa atgccgcgcg gatcctgaaa ggtatcttcc ggccaatgca
      721 gatcccacgg gcccatctgc acttcatcat acagcgggcc ccacgcgccg ctcggcggaa
      781 aggtgcgttc cacttcatca tacaaatgca tatggccctg aaaatacagg ttttcaaatt
      841 ctgaaatcct tccctcgatc ccgaggttgt tgttattgtt attgttgttg ttgttcgagc
      901 tcgaattagt ctgcgcgtct ttcagggctt catcgacagt ctgacgaccg ctggcggcgt
      961 tgatcaccgc agtacgcacg gcataccaga aagcggacat ctgcgggatg ttcggcatga
     1021 tttcaccttt ctgggcgttt tccatggtgg cggcaatacg tggatctttc gccaactctt
     1081 cctcgtaaga cttcagcgct acggcaccca gcggtttgtc tttattaacc gcttccagac
     1141 cttcatcagt cagcagatag ttttcgagga actcttttgc cagctctttg ttcggactgg
     1201 cggcgttaat acctgcgctc agcacgccaa cgaacggttt ggatggttga cccttgaagg
     1261 tcggcagtac cgttacacca taattcactt tgctggtgtc gatgttggac catgcccacg
     1321 ggccgttgat ggtcatcgct gtttcgcctt tattaaaggc agcttctgcg atggagtaat
     1381 cggtgtctgc attcatgtgt ttgtttttaa tcaggtcaac caggaaggtc agacccgctt
     1441 tcgcgccagc gttatccacg cccacgtctt taatgtcgta cttgccgttt tcatacttga
     1501 acgcataacc cccgtcagca gcaatcagcg gccaggtgaa gtacggttct tgcaggttga
     1561 acatcagcgc gctcttacct ttcgctttca gttctttatc cagcgccggg atctcttccc
     1621 aggtttttgg cgggttcggc agcagatctt tgttataaat cagcgataac gcttcaacag
     1681 cgatcgggta agcaatcagc ttgccgttgt aacgtacggc atcccaggta aacggataca
     1741 gcttgtcctg gaacgctttg tccggggtga tttcagccaa caggccagat tgagcgtagc
     1801 caccaaagcg gtcgtgtgcc cagaagataa tgtcagggcc atcgccagtt gccgcaacct
     1861 gtgggaattt ctcttccagt ttatccggat gctcaacggt gactttaatt ccggtatctt
     1921 tctcgaattt cttaccgact tcagcgagac cgttatagcc tttatcgccg ttaatccaga
     1981 ttaccagttt accttcttca gttttcataa tctatggtcc ttgttggtga agtgctcgtg
     2041 aaaacaccta aacggactgg ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2101 acattatacg agccgatgat taattgtcaa cagctcattt cagaatattt gccagaaccg
     2161 ttatgatgtc ggcgcaaaaa acattatcca gaacgggagt gcgccttgag cgacacgaat
     2221 tatgcagtga tttacgacct gcacagccat accacagctt ccgatggctg cctgacgcca
     2281 gaagcattgg tgcaccgtgc agtcgatgat aagctgtcaa acatgagaat tgtgcctaat
     2341 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     2401 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     2461 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     2521 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     2581 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     2641 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     2701 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     2761 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     2821 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     2881 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     2941 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     3001 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     3061 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     3121 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     3181 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     3241 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     3301 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     3361 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     3421 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     3481 tccgggcgct atcatgccat accgcgaaag gttttgcacc attcgatggt gtcggaattt
     3541 cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc
     3601 ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa
     3661 cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg
     3721 gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc
     3781 ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga
     3841 agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt
     3901 gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga
     3961 gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg
     4021 aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc
     4081 agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct
     4141 gtgaatcgct gcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg
     4201 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     4261 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     4321 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc
     4381 atcagagcag attgtactga gagtgcacca catgcggtgt actgagagtg caccacatgc
     4441 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     4501 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4561 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4621 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4681 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4741 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4801 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4861 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4921 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4981 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5041 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5101 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5161 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5221 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5281 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5341 tatcaaaaag gatcttcacc tagatccttt tacgcgccct gtagcggcgc attaagcgcg
     5401 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     5461 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5521 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5581 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     5641 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5701 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     5761 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     5821 acaatttaaa tatttgctta tacaatcttc ctgtttttgg ggcttttctg attatcaacc
     5881 ggggtaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5941 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6001 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6061 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6121 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6181 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6241 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6301 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6361 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6421 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6481 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6541 caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggraaac
     6601 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6661 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6721 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6781 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6841 gcggatacat atttgaatgt atttagaaaa ataaacaaaa gagtttgtag aaacgcaaaa
     6901 aggccatccg tcaggatggc cttc
//
