LOCUS       sequence-218943-        6009 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination; may not be full 
            ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6009)
  TITLE     SGC+year
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 6009)
  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: 
            "unpublished"
COMMENT     Sequence Label: MAGEA4
FEATURES             Location/Qualifiers
     source          1..6009
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             73..90
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             115..135
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             855..872
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(925..943)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      939..986
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1023..1478
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1110..1129)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1320..1341
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1570..2385)
                     /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(1570..2385)
                     /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     2293..2312
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2377..2396
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2507..3095
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2996..3015
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3249..3266
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3281..3420
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3346..3365)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3506..3528
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3522..3713)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4522..5604)
                     /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     5566..5585
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5605..5682)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5888..5907
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5991..6009,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5991..6009
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatatacata tgcaccatca tcatcatcat tcttctggtg tagatctggg taccgagaac
      121 ctgtacttcc aatccatgga cgcagagtcc ttgttccgag aagcactcag taacaaggtg
      181 gatgagttgg ctcattttct gctccgcaag tatcgagcca aggagctggt cacaaaggca
      241 gaaatgctgg agagagtcat caaaaattac aagcgctgct ttcctgtgat cttcggcaaa
      301 gcctccgagt ccctgaagat gatctttggc attgacgtga aggaagtgga ccccaccagc
      361 aacacctaca cccttgtcac ctgcctgggc ctttcctatg atggcctgct gggtaataat
      421 cagatctttc ccaagacagg ccttctgata atcgtcctgg gcacaattgc aatggagggc
      481 gacagcgcct ctgaggagga aatctgggag gagctgggtg tgatgggggt gtatgatggg
      541 agggagcaca ctgtctatgg ggagcccagg aaactgctca cccaagattg ggtgcaggaa
      601 aactacctgg agtaccggca ggtacccggc agtaatcctg cgcgctatga gttcctgtgg
      661 ggtccaaggg ctctggctga aaccagctat gtgaaagtcc tggagcatgt ggtcagggtc
      721 aatgcaagag ttcgcattgc ctacccatcc ctgcgtgaag cagctttgtt agaggaggaa
      781 gagggagtct gacagtaaag gtggatacgg atccgaattc gagctccgtc gacaagcttg
      841 cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
      901 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
      961 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     1021 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1081 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1141 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1201 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1261 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1321 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     1381 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     1441 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     1501 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1561 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     1621 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     1681 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     1741 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     1801 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     1861 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     1921 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     1981 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     2041 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     2101 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     2161 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     2221 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     2281 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     2341 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     2401 actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
     2461 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2521 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2581 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2641 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2701 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2761 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2821 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2881 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2941 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3001 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3061 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3121 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3181 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3241 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     3301 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     3361 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3421 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3481 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3541 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     3601 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     3661 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     3721 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     3781 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     3841 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     3901 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     3961 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     4021 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     4081 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     4141 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     4201 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4261 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     4321 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     4381 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     4441 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     4501 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4561 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4621 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4681 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4741 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     4801 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     4861 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     4921 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     4981 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5041 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5101 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5161 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5221 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5281 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5341 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5401 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5461 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5521 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5581 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     5641 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     5701 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     5761 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     5821 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     5881 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     5941 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     6001 cactatagg
//
