LOCUS       sequence-214886-        7272 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of EPHB4_Ephrin-lbd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7272)
  AUTHORS   Teyra J, Kelil A, Jain S, Helmy M, Jajodia R, Hooda Y, Gu J, D'Cruz 
            AA, Nicholson SE, Min J, Sudol M, Kim PM, Bader GD, Sidhu SS
  TITLE     Large-scale survey and database of high affinity ligands for peptide
            recognition modules.
  JOURNAL   Mol Syst Biol. 2020 Dec;16(12):e9310. doi: 10.15252/msb.20199310.
  PUBMED    33438817
REFERENCE   2  (bases 1 to 7272)
  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: "Mol Syst 
            Biol."; date: "2020-12"; volume: "16(12):e9310. doi"; pages: " 
            10.15252/msb.20199310"
COMMENT     Sequence Label: pHH0103_EPHB4_Ephrin-lbd
FEATURES             Location/Qualifiers
     source          1..7272
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             138..155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             171..824
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     782..804
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             831..854
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             867..884
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      1467..1514
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3107..3562)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3244..3265)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3456..3475
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3604..3626)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3712..3852
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3767..3786
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3867..3884)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4038..4625)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4796..5587,5588..5656))
                     /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     5419..5438
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5657..5761)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5829..5847
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5870..5947
                     /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             5948..7030
                     /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(5967..5986)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga
      121 ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt atgtccccta
      181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
      841 tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc gcggcccagc
      901 cggccgccct ggaagaaacc ctgctgaaca cgaaactgga aaccgcggat ctgaagtggg
      961 tcacctttcc gcaggtggac ggccaatggg aagaactgag tggtctggat gaagaacagc
     1021 attccgttcg tacgtatgaa gtttgcgatg tccagcgtgc accgggtcaa gcacattggc
     1081 tgcgtaccgg ttgggtcccg cgtcgcggtg cagtgcacgt ttacgctacg ctgcgcttca
     1141 ccatgctgga atgcctgagc ctgccgcgtg caggtcgttc ttgtaaagaa acctttacgg
     1201 tgttctatta cgaatctgat gcagacaccg caacggctct gaccccggct tggatggaaa
     1261 acccgtatat taaagtggac acggttgcgg ccgaacatct gacccgtaag cgtccgggtg
     1321 cagaagcaac gggtaaagtc aatgtgaaga ccctgcgtct gggcccgctg tcaaaagcgg
     1381 gtttttatct ggccttccag gatcaaggcg catgtatggc tctgctgtcg ctgcacctgt
     1441 tttacaaaaa ggcggccgca taataaaccg atacaattaa aggctccttt tggagccttt
     1501 tttttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc
     1561 tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aaccccatac agaaaattca
     1621 tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta tgagggctgt
     1681 ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg aaactcagtg tctagctaga
     1741 gtggcggtgg ctctggttcc ggtgattttg attatgaaaa gatggcaaac gctaataagg
     1801 gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc tgacgctaaa ggcaaacttg
     1861 attctgtcgc tactgattac ggtgctgcta tcgatggttt cattggtgac gtttccggcc
     1921 ttgctaatgg taatggtgct actggtgatt ttgctggctc taattcccaa atggctcaag
     1981 tcggtgacgg tgataattca cctttaatga ataatttccg tcaatattta ccttccctcc
     2041 ctcaatcggt tgaatgtcgc ccttttgtct ttggcgctgg taaaccatat gaattttcta
     2101 ttgattgtga caaaataaac ttattccgtg gtgtctttgc gtttctttta tatgttgcca
     2161 cctttatgta tgtattttct acgtttgcta acatactgcg taataaggag tcttaatcat
     2221 gccagttctt ttggctagcg ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg
     2281 tgcatggagc cgggccacct cgacctgaat ggaagccggc ggcacctcgc taacggattc
     2341 accactccaa gaattggagc caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac
     2401 ccttggcaga acatatccat cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg
     2461 ggcagcgttg ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg
     2521 ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg
     2581 tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt
     2641 ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg
     2701 atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag
     2761 cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt
     2821 ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc
     2881 atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag
     2941 gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag
     3001 acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt
     3061 gaatcgcttc acgaccacgc tgatgagctt taccgcagga tccgcgaaat tgtaagcgtt
     3121 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     3181 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     3241 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     3301 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     3361 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     3421 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     3481 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     3541 aatgcgccgc tacagggcgc gtcgcggatc ctgcctcgcg cgtttcggtg atgacggtga
     3601 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     3661 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     3721 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     3781 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     3841 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3901 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3961 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4021 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4081 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggg tttccccctg
     4141 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4201 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4261 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4321 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4381 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4441 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     4501 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4561 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4621 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4681 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4741 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     4801 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     4861 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     4921 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     4981 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5041 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5101 ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5161 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5221 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5281 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5341 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5401 gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     5461 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5521 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5581 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5641 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5701 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5761 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     5821 cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattcccg acaccatcga
     5881 atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
     5941 ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
     6001 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
     6061 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
     6121 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
     6181 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
     6241 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
     6301 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
     6361 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
     6421 tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
     6481 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
     6541 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
     6601 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
     6661 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
     6721 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
     6781 cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     6841 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     6901 gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
     6961 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     7021 cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca caattctcat
     7081 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
     7141 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
     7201 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
     7261 tgaaatgagc tg
//
