LOCUS       sequence-260921-        5402 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5402)
  TITLE     Top7 plasmids
REFERENCE   2  (bases 1 to 5402)
  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 H1-H2
FEATURES             Location/Qualifiers
     source          1..5402
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     119..138
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     226..245
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        226..244
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    245..269
                     /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             284..306
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             488..505
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(558..576)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      572..619
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      656..1111
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(743..762)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     953..974
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1203..2018)
                     /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(1203..2018)
                     /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     1926..1945
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2010..2029
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2140..2728
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2629..2648
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2882..2899
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2914..3053
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2979..2998)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3139..3161
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3155..3346)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4155..5237)
                     /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     5199..5218
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5238..5315)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
       61 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
      121 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
      181 tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaattaata cgactcacta
      241 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
      301 aggagatata catatggaag gtgcatcgga attacagaag gttttgaacg aactgaaaga
      361 ctacatcaaa aaacaggggg ctaaacgcgt tcgcatctcg attaccgctc gtacgaaaaa
      421 agaagcagaa aaatttgccg cgattttgat taaagttttc gccgaactgg gttataatgg
      481 tctcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
      541 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
      601 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
      661 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
      721 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
      781 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
      841 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
      901 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
      961 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     1021 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     1081 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     1141 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     1201 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     1261 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     1321 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     1381 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     1441 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     1501 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     1561 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     1621 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     1681 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     1741 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     1801 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     1861 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     1921 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     1981 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     2041 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     2101 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2161 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2221 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2281 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2341 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2401 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2461 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2521 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2581 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2641 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2701 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2761 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2821 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2881 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2941 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     3001 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3061 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3121 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3181 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     3241 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     3301 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     3361 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     3421 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     3481 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     3541 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     3601 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     3661 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     3721 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     3781 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     3841 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     3901 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     3961 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     4021 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     4081 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     4141 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4201 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     4261 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     4321 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     4381 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
     4441 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     4501 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     4561 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     4621 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     4681 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     4741 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     4801 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     4861 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     4921 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     4981 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     5041 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     5101 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     5161 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     5221 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     5281 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     5341 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     5401 aa
//
