LOCUS       sequence-330609-        5613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human dmU1A with the F37M/F77M double mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5613)
  AUTHORS   Srivastava Y, Bonn-Breach R, Chavali SS, Lippa GM, Jenkins JL, 
            Wedekind JE
  TITLE     Affinity and Structural Analysis of the U1A RNA Recognition Motif 
            with Engineered Methionines to Improve Experimental Phasing.
  JOURNAL   Crystals (Basel). 2021 Mar;11(3). doi: 10.3390/cryst11030273. Epub 
            2021 Mar 10.
  PUBMED    33777416
REFERENCE   2  (bases 1 to 5613)
  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: "Crystals 
            (Basel)."; date: "2021-03"; volume: "11(3). doi"; pages: " 
            10.3390/cryst11030273. Epub 2021 Mar 10"
COMMENT     Sequence Label: pET28a-TEV-U1AF37MF77M
FEATURES             Location/Qualifiers
     source          1..5613
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     946..965
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(985..1062)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1268..1287
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1371..1390
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1371..1389
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1390..1414
                     /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             1429..1451
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1470..1487
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1497..1517
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1848..1865
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1918..1936)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1932..1979
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2016..2471
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2103..2122)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2313..2334
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2563..3378)
                     /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             complement(2563..3378)
                     /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     3286..3305
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3370..3389
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3500..4088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3989..4008
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4242..4259
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4274..4413
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4339..4358)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4499..4521
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4515..4706)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5515..5613,1..984))
                     /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"
ORIGIN
        1 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
       61 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
      121 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
      181 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
      241 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
      301 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
      361 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
      421 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
      481 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
      541 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
      601 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
      661 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
      721 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
      781 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
      841 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
      901 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
      961 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     1021 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     1081 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     1141 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     1201 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     1261 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     1321 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     1381 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
     1441 taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc agcggcgaga
     1501 atctttattt tcagggccat atggcagttc ccgagacccg ccctaaccac actatttata
     1561 tcaacaacct caatgagaag atcaagaagg atgagctaaa aaagtccctg cacgccatct
     1621 tctcccggat gggccagatc ctggatatcc tggtatcacg gagcctgaag atgaggggcc
     1681 aggcctttgt catcttcaag gaggtcagca gcgccaccaa cgccctgcgc tccatgcagg
     1741 gtttccctat gtatgacaaa cctatgcgta tccagtatgc caagaccgac tcagatatca
     1801 ttgccaagat gaaatgatag gtcgacaagc ttgcggccgc actcgagcac caccaccacc
     1861 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     1921 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     1981 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     2041 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2101 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2161 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2221 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2281 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2341 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2401 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2461 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2521 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     2581 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     2641 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     2701 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     2761 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     2821 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     2881 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     2941 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     3001 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     3061 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     3121 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     3181 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     3241 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     3301 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     3361 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     3421 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3481 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3541 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3601 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     3661 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3721 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3781 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3841 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3901 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3961 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4021 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4081 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4141 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4201 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4261 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4321 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4381 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4441 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4501 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     4561 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     4621 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     4681 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     4741 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     4801 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     4861 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     4921 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     4981 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     5041 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     5101 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     5161 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     5221 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     5281 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     5341 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     5401 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     5461 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     5521 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5581 gggagaggcg gtttgcgtat tgggcgccag ggt
//
