LOCUS       sequence-331056-        6419 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 6419)
  TITLE     SGC Toronto Q3-Q4 2020
REFERENCE   2  (bases 1 to 6419)
  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"
COMMENT     Sequence Label: FEM1C
FEATURES             Location/Qualifiers
     source          1..6419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     28..47
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        28..46
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    47..71
                     /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             86..108
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             118..135
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             148..168
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1311..1328
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1381..1399)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1395..1442
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1479..1934
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1566..1585)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1776..1797
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2026..2841)
                     /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(2026..2841)
                     /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     2749..2768
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2833..2852
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2963..3551
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3452..3471
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3705..3722
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3737..3876
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3802..3821)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3962..3984
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3978..4169)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4978..6060)
                     /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     6022..6041
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6061..6138)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6344..6363
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
       61 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgcat
      121 catcatcatc atcacagcag cggcagagaa aacttgtatt tccagggcat ggatctaaag
      181 acagcagtat ttaacgcagc tcgggatggc aaactccggc ttctcaccaa attgttggca
      241 agcaaatcca aagaggaggt ttcctccttg atctctgaaa aaacaaatgg ggccacgcca
      301 ctcttgatgg ccgccaggta tgggcacctt gacatggtgg aattcctcct agagcaatgc
      361 agtgcctcca tagaagttgg gggctccgtc aattttgatg gcgaaaccat tgagggggct
      421 ccccctttat gggccgcttc tgcagcagga catctgaagg tggtccagtc cttgttaaat
      481 catggagcat ctgtcaacaa cacgacttta accaattcaa ctcctcttcg agctgcgtgt
      541 ttcgatggcc atttggaaat agtgaagtac cttgtagaac acaaagctga tttggaagtg
      601 tcaaaccgac atgggcatac gtgcttgatg atttcatgtt acaaaggaca taaagagatt
      661 gctcagtatt tacttgaaaa gggggcagat gttaatagaa aaagtgtcaa aggtaatact
      721 gcattgcatg attgtgcaga atctggaagt ttggacatca tgaagatgct tcttatgtat
      781 tgtgccaaga tggaaaagga tggttatgga atgactcccc ttctctcagc aagtgtgact
      841 ggtcacacaa atattgtgga ttttctgaca caccatgcac agaccagcaa gacagaacgt
      901 attaatgctc tagagcttct gggagctaca tttgtagaca aaaaaagaga tctgcttggg
      961 gctttgaaat actggaaaaa ggcaatgaac atgaggtaca gtgataggac taatattatt
     1021 agtaaaccag tgccacagac actaataatg gcttatgatt atgccaagga agtgaacagt
     1081 gcagaagagc tagaaggtct tattgctgat cctgatgaga tgagaatgca ggcactatta
     1141 atcagagaac gtattcttgg tccttctcat cctgatacct cttactatat tagatataga
     1201 ggcgctgtct atgcagactc tggaaatttc aaacgatgca tcaacctatg gaagtatgct
     1261 ttggatatgc agcagagcaa ttgatgacga agcttgcggc cgcactcgag caccaccacc
     1321 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     1381 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     1441 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     1501 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     1561 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     1621 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     1681 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     1741 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     1801 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     1861 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     1921 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     1981 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     2041 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     2101 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     2161 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     2221 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     2281 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     2341 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     2401 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     2461 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     2521 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     2581 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     2641 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     2701 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     2761 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     2821 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     2881 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2941 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3001 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3061 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3121 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3181 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3241 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3301 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3361 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3421 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3481 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3541 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3601 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3661 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3721 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3781 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     3841 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3901 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3961 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     4021 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     4081 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     4141 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     4201 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     4261 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     4321 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     4381 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     4441 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     4501 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     4561 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     4621 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     4681 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     4741 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     4801 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     4861 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     4921 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     4981 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5041 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     5101 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     5161 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     5221 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     5281 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     5341 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     5401 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     5461 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     5521 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     5581 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     5641 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     5701 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     5761 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     5821 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     5881 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     5941 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     6001 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     6061 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     6121 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     6181 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     6241 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     6301 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     6361 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtag
//
