LOCUS       sequence-357182-        6249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human fibronectin fragment, type-III repeats 9-10, R1374A, P1376A, 
            R1379A mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6249)
  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 6249)
  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: FnRPR9-10
FEATURES             Location/Qualifiers
     source          1..6249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      390..978
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     879..898
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1132..1149
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1164..1303
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1229..1248)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1389..1411
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1405..1596)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2908..3990)
                     /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     3952..3971
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3991..4068)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4274..4293
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4377..4396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4377..4395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4396..4420
                     /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             4435..4457
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4468..4494
                     /codon_start=1
                     /product="9xHis affinity tag"
                     /label=9xHis
                     /translation="HHHHHHHHH"
     CDS             4495..4515
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(5126..5144)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5140..5187
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5362..5367,5368..5384,5385..5390))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5417..5435)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5503..5607
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5608..5676,5677..6249,1..219)
                     /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(5826..5845)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
       61 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
      121 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
      181 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
      241 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
      301 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
      361 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
      421 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
      481 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
      541 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
      601 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
      661 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
      721 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
      781 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
      841 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
      901 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
      961 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     1021 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     1081 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     1141 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     1201 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     1261 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     1321 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     1381 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     1441 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     1501 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     1561 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     1621 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     1681 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     1741 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     1801 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     1861 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     1921 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     1981 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     2041 gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg
     2101 gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac
     2161 agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg
     2221 ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg
     2281 aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc
     2341 gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag gctggtaaga
     2401 gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg
     2461 gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc
     2521 atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg
     2581 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     2641 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     2701 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     2761 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     2821 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     2881 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     2941 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3001 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     3061 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     3121 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     3181 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     3241 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     3301 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     3361 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     3421 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     3481 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     3541 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     3601 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     3661 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     3721 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     3781 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     3841 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     3901 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     3961 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     4021 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     4081 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     4141 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     4201 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     4261 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     4321 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
     4381 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     4441 taactttaag aaggagatat acatatgcac caccaccacc atcatcacca tcacgaaaac
     4501 ttatacttcc agggtacagg tcttgattcc ccaactggca ttgacttttc tgatattact
     4561 gccaactctt ttactgtgca ctggattgct cctcgagcca ccatcactgg ctacaggatc
     4621 cgccatcatc ccgagcactt cagtgggaga cctcgagaag atgcggtggc gcactctgcg
     4681 aattccatca ccctcaccaa cctcactcca ggcacagagt atgtggtcag catcgttgct
     4741 cttaatggca gagaggaaag tcccttattg attggccaac aatcaacagt ttctgatgtt
     4801 ccgagggacc tggaagttgt tgctgcgacc cccaccagcc tactgatcag ctgggatgct
     4861 cctgctgtca cagtgagata ttacaggatc acttacggag agacaggagg aaatagccct
     4921 gtccaggagt tcactgtgcc tgggagcaag tctacagcta ccatcagcgg ccttaaacct
     4981 ggagttgatt ataccatcac tgtgtatgct gtcactggcc gtggagacag ccccgcaagc
     5041 agcaagccaa tttccattaa ttaccgaaca tgataggatc cggctgctaa caaagcccga
     5101 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     5161 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatcccgca
     5221 agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc
     5281 cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat
     5341 ttaactgtga taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat
     5401 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5461 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5521 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5581 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5641 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5701 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5761 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5821 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     5881 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5941 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6001 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6061 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6121 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     6181 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6241 gataaagtt
//
