LOCUS       sequence-258528-        6306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of ETS gene SPDEF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6306)
  AUTHORS   Selvaraj N, Kedage V, Hollenhorst PC
  TITLE     Comparison of MAPK specificity across the ETS transcription factor 
            family identifies a high-affinity ERK interaction required for ERG 
            function in prostate cells.
  JOURNAL   Cell Commun Signal. 2015 Feb 19;13:12. doi: 
            10.1186/s12964-015-0089-7.
  PUBMED    25885538
REFERENCE   2  (bases 1 to 6306)
  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: "Cell Commun
            Signal."; date: "2015-02-19"; volume: "13:12. doi"; pages: " 
            10.1186/s12964-015-0089-7"
COMMENT     Sequence Label: SPDEF_pet28a
FEATURES             Location/Qualifiers
     source          1..6306
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1184..1201)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1211..1228)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1247..1269
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1284..1308
                     /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     complement(1308..1327)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1309..1327)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1411..1430)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1636..1713
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1714..2796
                     /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(1733..1752)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3605..3796
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3790..3812)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3898..4037
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3953..3972
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4052..4069)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4223..4811)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4303..4322)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4922..4941)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4933..5748
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4933..5748
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5006..5025)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5840..6295)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5977..5998)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6189..6208
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gctcagatgg
      181 ggtgcacgaa ctggtagacg aggcgctggg agatgtctgg cttccggatg atgcccttct
      241 tgtaatactg gcggatggag cggctcagct tgtcgtagtt catggcggga cggttcttgc
      301 ggatgcccca cagccgggcc acctgggctg agtcctcaat tttgaagatg cccttctcct
      361 tgttgagcca cctaatgaag cggccatagc tgtggggctt gagtagcaac tccttgagga
      421 actgccacag gtggatgggc tgcccggagc atgatgagtc cacctcgctg tcggtccagc
      481 tctcctcact ggtcgaggca cagtagtgaa tcgccccagg tgaagtccgc tctttcatcc
      541 aggccgctga cttccagatg tccaggtggg cgtgcagcac atccccaccc aggggcgagc
      601 gctggcggaa ctgctcctcc gacatggcgc acagctcctt gcccgccagc tcctggaagg
      661 ccttgcccat ggggggcagc cggtattggt gctctgtcca caggagccac ttctgcacat
      721 tgctggggct ccagtccatg ggatctgcgg tgatgttgag cagcttgcag gccgtctcga
      781 tgtccttgag cacttcgccc accaccatgg actgcacctg ctccagcgag tgctcctcca
      841 aggtcagccc gccgggcacc aagtccaggc tgcccgctgg ggcttggctg tcaatgaccg
      901 ggcactgctc aggctcctca ggtggctcct cccgactgct ggccccaggg gccttggctg
      961 cccagctgct gtcctcaggg tacagcatgt caaagtagga gaggtagaag gcggacaggc
     1021 cctgctcggg cgtggcgggt ggactgggac tccagtcccg tctctcgaga cccactgccc
     1081 ccgctgccgc cttctccaag cctgtccgcg acaccgtgtc ggggggcagc aggaggtggc
     1141 tgggggatac gctgctcaga cccgggctgg cgctgcccat atggctgccg cgcggcacca
     1201 ggccgctgct gtgatgatga tgatgatggc tgctgcccat ggtatatctc cttcttaaag
     1261 ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt
     1321 cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc gtggccggca
     1381 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag
     1441 atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc
     1501 ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg
     1561 tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag
     1621 agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag
     1681 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
     1741 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
     1801 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
     1861 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
     1921 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
     1981 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
     2041 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
     2101 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
     2161 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
     2221 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
     2281 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
     2341 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
     2401 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
     2461 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
     2521 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
     2581 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     2641 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     2701 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     2761 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
     2821 gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag
     2881 ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat
     2941 catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt
     3001 tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct
     3061 cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat
     3121 cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct
     3181 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg
     3241 aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt
     3301 ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat
     3361 ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg
     3421 ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt
     3481 accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat
     3541 cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc
     3601 atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac
     3661 attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga
     3721 atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga
     3781 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     3841 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     3901 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     3961 gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga
     4021 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4081 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4141 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4201 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4261 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4321 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4381 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4441 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4501 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4561 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4621 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     4681 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4741 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4801 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4861 actcacgtta agggattttg gtcatgaaca ataaaactgt ctgcttacat aaacagtaat
     4921 acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat
     4981 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     5041 ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     5101 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     5161 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     5221 ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca
     5281 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     5341 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     5401 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     5461 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     5521 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     5581 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     5641 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     5701 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga attaattcat
     5761 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5821 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     5881 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     5941 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     6001 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     6061 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     6121 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     6181 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     6241 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     6301 tcgcca
//
