LOCUS       sequence-192614-        6927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            RAB4B-2/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6927)
  AUTHORS   Teyra J, Huang H, Jain S, Guan X, Dong A, Liu Y, Tempel W, Min J, 
            Tong Y, Kim PM, Bader GD, Sidhu SS
  TITLE     Comprehensive Analysis of the Human SH3 Domain Family Reveals a Wide
            Variety of Non-canonical Specificities.
  JOURNAL   Structure. 2017 Oct 3;25(10):1598-1610.e3. doi: 
            10.1016/j.str.2017.07.017. Epub 2017 Sep 7.
  PUBMED    28890361
REFERENCE   2  (bases 1 to 6927)
  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"; doi: 
            "10.1016/j.str.2017.07.017"; journalName: "Structure"; date: 
            "2017-10-3- 3"; volume: "25"; issue: "10"; pages: "1598-1610.e3"
COMMENT     Sequence Label: pHH0103_RAB4B-2/2
FEATURES             Location/Qualifiers
     source          1..6927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..47
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        70..147
                     /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             148..1230
                     /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(167..186)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1473..1478,1479..1494,1495..1501)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1509..1525
                     /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     1514..1536
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1610..1627
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1643..2296
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2254..2276
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2303..2326
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2339..2356
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2594..2641
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4234..4689)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4371..4392)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4583..4602
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4731..4753)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4839..4979
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4894..4913
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4994..5011)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5165..5752)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5923..6714,6715..6783))
                     /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     6546..6565
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6784..6888)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca
       61 agaattcccg acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg
      121 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
      181 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
      241 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
      301 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
      361 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
      421 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
      481 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
      541 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
      601 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
      661 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
      721 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
      781 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
      841 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
      901 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
      961 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     1021 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     1081 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     1141 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     1201 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     1261 tcattaggca caattctcat gtttgacagc ttatcatcga ctgcacggtg caccaatgct
     1321 tctggcgtca ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca
     1381 taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat
     1441 aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatcgg ctcgtataat
     1501 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agccagtccg tttaggtgtt
     1561 ttcacgagca cttcaccaac aaggaccata gattatgaaa atcgaagaac accatcacca
     1621 tcaccattcc agcggtaagc ttatgtcccc tatactaggt tattggaaaa ttaagggcct
     1681 tgtgcaaccc actcgacttc ttttggaata tcttgaagaa aaatatgaag agcatttgta
     1741 tgagcgcgat gaaggtgata aatggcgaaa caaaaagttt gaattgggtt tggagtttcc
     1801 caatcttcct tattatattg atggtgatgt taaattaaca cagtctatgg ccatcatacg
     1861 ttatatagct gacaagcaca acatgttggg tggttgtcca aaagagcgtg cagagatttc
     1921 aatgcttgaa ggagcggttt tggatattag atacggtgtt tcgagaattg catatagtaa
     1981 agactttgaa actctcaaag ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt
     2041 cgaagatcgt ttatgtcata aaacatattt aaatggtgat catgtaaccc atcctgactt
     2101 catgttgtat gacgctcttg atgttgtttt atacatggac ccaatgtgcc tggatgcgtt
     2161 cccaaaatta gtttgtttta aaaaacgtat tgaagctatc ccacaaattg ataagtactt
     2221 gaaatccagc aagtatatag catggccttt gcagggctgg caagccacgt ttggtggtgg
     2281 cgaccatcct ccaaaatcgg atctggaagt tctgttccag gggcccctgt ccagcggtct
     2341 ggttccgcgt ggttccggta ccgcggccca gccggcctct atggcagttg ccctgcaaga
     2401 ttacatggca ccggattgtc gttttctgac cattcatcgt ggtcaggtcg tctacgtttt
     2461 ctccaaactg aaaggtcgtg gtcgtttgtt ttggggtggt agtgttcagg gtgattacta
     2521 tggtgatctg gcagcacgtc ttggttactt tccgtcctcc attgttcgtg aagatcaggc
     2581 ggccgcataa taaaccgata caattaaagg ctccttttgg agcctttttt ttggagattt
     2641 tcaacgtgaa aaaattatta ttcgcaattc ctttagttgt tcctttctat tctcactccg
     2701 ctgaaactgt tgaaagttgt ttagcaaaac cccatacaga aaattcattt actaacgtct
     2761 ggaaagacga caaaacttta gatcgttacg ctaactatga gggctgtctg tggaatgcta
     2821 caggcgttgt agtttgtact ggtgacgaaa ctcagtgtct agctagagtg gcggtggctc
     2881 tggttccggt gattttgatt atgaaaagat ggcaaacgct aataaggggg ctatgaccga
     2941 aaatgccgat gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt ctgtcgctac
     3001 tgattacggt gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa
     3061 tggtgctact ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga
     3121 taattcacct ttaatgaata atttccgtca atatttacct tccctccctc aatcggttga
     3181 atgtcgccct tttgtctttg gcgctggtaa accatatgaa ttttctattg attgtgacaa
     3241 aataaactta ttccgtggtg tctttgcgtt tcttttatat gttgccacct ttatgtatgt
     3301 attttctacg tttgctaaca tactgcgtaa taaggagtct taatcatgcc agttcttttg
     3361 gctagcgccg ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg
     3421 gccacctcga cctgaatgga agccggcggc acctcgctaa cggattcacc actccaagaa
     3481 ttggagccaa tcaattcttg cggagaactg tgaatgcgca aaccaaccct tggcagaaca
     3541 tatccatcgc gtccgccatc tccagcagcc gcacgcggcg catctcgggc agcgttgggt
     3601 cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg
     3661 gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct
     3721 gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt
     3781 aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag
     3841 gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac
     3901 cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac
     3961 gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt
     4021 tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca
     4081 aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc
     4141 tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg
     4201 accacgctga tgagctttac cgcaggatcc gcgaaattgt aagcgttaat attttgttaa
     4261 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     4321 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     4381 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     4441 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     4501 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     4561 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     4621 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     4681 agggcgcgtc gcggatcctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     4741 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     4801 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     4861 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     4921 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     4981 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5041 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5101 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5161 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5221 ggtggcgaaa cccgacagga ctataaagat accagggttt ccccctggaa gctccctcgt
     5281 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5341 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5401 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5461 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5521 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5581 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     5641 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5701 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5761 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5821 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5881 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5941 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6001 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6061 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6121 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6181 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc
     6241 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6301 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6361 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6421 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6481 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac
     6541 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6601 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6661 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6721 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6781 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6841 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6901 aaaagtgcca cctgacgtct aagaaac
//
