LOCUS       sequence-357184-        6516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human fibronectin fragment, type-III repeats 8-10, R1374A, P1376A, 
            R1379A mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6516)
  AUTHORS   Su Y, Iacob RE, Li J, Engen JR, Springer TA
  TITLE     Dynamics of integrin alpha5beta1, fibronectin, and their complex 
            reveal sites of interaction and conformational change.
  JOURNAL   J Biol Chem. 2022 Sep;298(9):102323. doi: 10.1016/j.jbc.2022.102323.
            Epub 2022 Aug 2.
  PUBMED    35931112
REFERENCE   2  (bases 1 to 6516)
  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: "J Biol 
            Chem."; date: "2022-09"; volume: "298(9):102323. doi"; pages: " 
            10.1016/j.jbc.2022.102323. Epub 2022 Aug 2"
COMMENT     Sequence Label: FnRPR8-10
FEATURES             Location/Qualifiers
     source          1..6516
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(18..1100)
                     /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     1062..1081
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1101..1178)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1384..1403
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1487..1506
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1487..1505
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1506..1530
                     /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             1545..1567
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1578..1604
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     CDS             1605..1625
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2503..2521)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2517..2564
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2739..2744,2745..2761,2762..2767))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2794..2812)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2880..2984
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2985..3053,3054..3845)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3203..3222)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4016..4604
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4505..4524
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4758..4775
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4790..4929
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4855..4874)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5015..5037
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5031..5222)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
       61 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
      121 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
      181 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
      241 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
      301 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
      361 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
      421 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
      481 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
      541 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
      601 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
      661 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
      721 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
      781 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
      841 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
      901 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
      961 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     1021 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     1081 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     1141 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     1201 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     1261 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     1321 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     1381 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     1441 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
     1501 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
     1561 aaggagatat acatatgcac caccaccacc atcatcacca tcacgaaaac ttatacttcc
     1621 agggtgttcc tcctcccact gacctgcgat tcaccaacat tggtccagac accatgcgtg
     1681 tcacctgggc tccaccccca tccattgatt taaccaactt cctggtgcgt tactcacctg
     1741 tgaaaaatga ggaagatgtt gcagagttgt caatttctcc ttcagacaat gcagtggtct
     1801 taacaaatct cctgcctggt acagaatatg tagtgagtgt ctccagtgtc tacgaacaac
     1861 atgagagcac acctcttaga ggaagacaga aaacaggtct tgattcccca actggcattg
     1921 acttttctga tattactgcc aactctttta ctgtgcactg gattgctcct cgagccacca
     1981 tcactggcta caggatccgc catcatcccg agcacttcag tgggagacct cgagaagatg
     2041 cggtggcgca ctctgcgaat tccatcaccc tcaccaacct cactccaggc acagagtatg
     2101 tggtcagcat cgttgctctt aatggcagag aggaaagtcc cttattgatt ggccaacaat
     2161 caacagtttc tgatgttccg agggacctgg aagttgttgc tgcgaccccc accagcctac
     2221 tgatcagctg ggatgctcct gctgtcacag tgagatatta caggatcact tacggagaga
     2281 caggaggaaa tagccctgtc caggagttca ctgtgcctgg gagcaagtct acagctacca
     2341 tcagcggcct taaacctgga gttgattata ccatcactgt gtatgctgtc actggccgtg
     2401 gagacagccc cgcaagcagc aagccaattt ccattaatta ccgaacatga taggatccgg
     2461 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     2521 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     2581 tatccggata tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc
     2641 atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca tacacggtgc
     2701 ctgactgcgt tagcaattta actgtgataa actaccgcat taaagcttat cgatgataag
     2761 ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     2821 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     2881 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2941 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3001 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3061 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3121 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3181 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     3241 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3301 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3361 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3421 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3481 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     3541 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3601 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3661 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3721 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3781 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3841 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3901 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3961 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4021 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4081 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4141 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     4201 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4261 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4321 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4381 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4441 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4501 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4561 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4621 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4681 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4741 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     4801 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     4861 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     4921 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4981 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5041 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     5101 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     5161 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     5221 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     5281 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     5341 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     5401 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     5461 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     5521 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     5581 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     5641 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca
     5701 acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat ggatatgttc
     5761 tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc tccaattctt
     5821 ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag gtggcccggc
     5881 tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg cctacaatcc
     5941 atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg atcagcggtc
     6001 caatgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt
     6061 cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag
     6121 cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt
     6181 agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     6241 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     6301 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     6361 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     6421 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     6481 gtcgagatcc cggtgcctaa tgagtgagct aactta
//
