LOCUS       sequence-235394-        5528 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid contains Top7 K39E K40E V46A and K55E. Please note this is 
            DIFFERENT than what the plasmid name implies..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5528)
  TITLE     Top7 plasmids
REFERENCE   2  (bases 1 to 5528)
  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: Top7 K39E K40E V48V K55E
FEATURES             Location/Qualifiers
     source          1..5528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     103..122
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     210..229
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        210..228
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    229..253
                     /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             268..290
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             598..615
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(668..686)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      682..729
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      766..1221
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(853..872)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1063..1084
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1313..2128)
                     /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(1313..2128)
                     /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     2036..2055
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2120..2139
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2250..2838
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2739..2758
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2992..3009
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3024..3163
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3089..3108)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3249..3271
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3265..3456)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4265..5347)
                     /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     5309..5328
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5348..5425)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
       61 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
      121 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
      181 gatcgagatc gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
      241 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg
      301 ggggatatcc aggttcaagt gaacattgac gataatggta aaaacttcga ttatacttat
      361 accgtgacca cagaatcgga attacagaag gttttgaacg aactgaaaga ctacatcgaa
      421 gaacaggggg ctaaacgcgc gcgcatctcg attaccgctc gtacggaaaa agaagcagaa
      481 aaatttgccg cgattttgat taaagttttc gccgaactgg gttataatga tattaacgtt
      541 acgtgggacg gcgatacggt gacagtggaa ggacagttag agggcggttc tctcgagcac
      601 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
      661 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
      721 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
      781 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
      841 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
      901 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
      961 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1021 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     1081 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     1141 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     1201 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1261 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     1321 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     1381 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     1441 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     1501 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     1561 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     1621 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     1681 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     1741 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     1801 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     1861 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     1921 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     1981 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     2041 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     2101 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     2161 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2221 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2281 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2341 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     2401 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2461 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2521 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     2581 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2641 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     2701 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2761 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2821 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     2881 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     2941 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3001 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3061 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3121 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     3181 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3241 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3301 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     3361 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     3421 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     3481 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     3541 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     3601 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     3661 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     3721 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     3781 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     3841 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     3901 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     3961 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     4021 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     4081 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     4141 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     4201 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     4261 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4321 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     4381 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     4441 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     4501 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
     4561 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     4621 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     4681 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     4741 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     4801 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     4861 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     4921 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     4981 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     5041 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     5101 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     5161 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     5221 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     5281 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     5341 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     5401 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     5461 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     5521 gcatgcaa
//
