LOCUS       Exported                5368 bp ds-DNA     circular SYN 13-SEP-2025
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    pET-28b(+)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5368)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5368)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 13, 2025 from SnapGene 4.1.9
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5368
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          join(485..5368,1..484)
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        100..118
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    119..143
                     /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             158..180
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             199..216
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             226..243
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             247..279
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             328..345
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      412..459
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      496..951
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(1044..1859)
                     /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"
     rep_origin      1981..2569
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    2755..2897
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2999..3190)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3999..5081)
                     /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"
     promoter        5082..5159
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
       61 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
      121 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
      181 tataccatgg gcagcagcca tcatcatcat catcacagca gcggcctggt gccgcgcggc
      241 agccatatgg ctagcatgac tggtggacag caaatgggtc gggatccgaa ttcgagctcc
      301 gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct
      361 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
      421 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
      481 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
      541 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
      601 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      661 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
      721 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
      781 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
      841 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
      901 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tcaggtggca
      961 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     1021 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     1081 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     1141 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     1201 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     1261 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     1321 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     1381 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     1441 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     1501 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     1561 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     1621 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     1681 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     1741 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     1801 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     1861 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     1921 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     1981 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2041 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2101 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     2161 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2221 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2281 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2341 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2401 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2461 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     2521 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     2581 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     2641 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     2701 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     2761 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc
     2821 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     2881 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     2941 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     3001 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     3061 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     3121 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     3181 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     3241 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     3301 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     3361 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     3421 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     3481 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     3541 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     3601 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     3661 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     3721 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     3781 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     3841 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     3901 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     3961 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4021 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4081 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     4141 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     4201 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     4261 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4321 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     4381 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     4441 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     4501 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     4561 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     4621 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     4681 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     4741 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     4801 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     4861 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     4921 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     4981 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     5041 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     5101 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     5161 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     5221 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     5281 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     5341 gtggcgagcc cgatcttccc catcggtg
//
