LOCUS       sequence-228306-        6210 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 6210)
  TITLE     other plasmids
REFERENCE   2  (bases 1 to 6210)
  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: BirA in pET28a (w400-2)
FEATURES             Location/Qualifiers
     source          1..6210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     98..115
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    130..269
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(195..214)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     355..377
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(371..562)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1371..2453)
                     /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     2415..2434
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2454..2531)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2737..2756
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2840..2859
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2840..2858
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2859..2883
                     /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             2898..2920
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2927..3889
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             2927..3889
                     /codon_start=1
                     /gene="birA"
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     CDS             3914..3931
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3984..4002)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3998..4045
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4082..4537
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4169..4188)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4379..4400
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4629..5444)
                     /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(4629..5444)
                     /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     5352..5371
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5436..5455
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5566..6154
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6055..6074
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
       61 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
      121 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
      181 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
      241 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
      301 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
      361 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
      421 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
      481 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
      541 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
      601 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
      661 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
      721 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
      781 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
      841 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
      901 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
      961 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     1021 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     1081 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     1141 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     1201 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     1261 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     1321 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     1381 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     1441 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     1501 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     1561 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     1621 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     1681 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     1741 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     1801 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     1861 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     1921 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     1981 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     2041 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     2101 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     2161 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     2221 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     2281 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     2341 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     2401 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     2461 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     2521 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     2581 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     2641 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     2701 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     2761 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     2821 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     2881 tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatga aggataacac
     2941 cgtgccactg aaattgattg ccctgttagc gaacggtgaa tttcactctg gcgagcagtt
     3001 gggtgaaacg ctgggaatga gccgggcggc tattaataaa cacattcaga cactgcgtga
     3061 ctggggcgtt gatgtcttta ccgttccggg taaaggatac agcctgcctg agcctatcca
     3121 gttacttaat gctaaacaga tattgggtca gctggatggc ggtagtgtag ccgtgctgcc
     3181 agtgattgac tccacgaatc agtaccttct tgatcgtatc ggagagctta aatcgggcga
     3241 tgcttgcatt gcagaatacc agcaggctgg ccgtggtcgc cggggtcgga aatggttttc
     3301 gccttttggc gcaaacttat atttgtcgat gttctggcgt ctggaacaag gcccggcggc
     3361 ggcgattggt ttaagtctgg ttatcggtat cgtgatggcg gaagtattac gcaagctggg
     3421 tgcagataaa gttcgtgtta aatggcctaa tgacctctat ctgcaggatc gcaagctggc
     3481 aggcattctg gtggagctga ctggcaaaac tggcgatgcg gcgcaaatag tcattggagc
     3541 cgggatcaac atggcaatgc gccgtgttga agagagtgtc gttaatcagg ggtggatcac
     3601 gctgcaggaa gcggggatca atctcgatcg taatacgttg gcggccatgc taatacgtga
     3661 attacgtgct gcgttggaac tcttcgaaca agaaggattg gcaccttatc tgtcgcgctg
     3721 ggaaaagctg gataatttta ttaatcgccc agtgaaactt atcattggtg ataaagaaat
     3781 atttggcatt tcacgcggaa tagacaaaca gggggcttta ttacttgagc aggatggaat
     3841 aataaaaccc tggatgggcg gtgaaatatc cctgcgtagt gcagaaaaag gtaagcttgc
     3901 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     3961 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     4021 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     4081 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4141 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4201 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4261 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     4321 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4381 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4441 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4501 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     4561 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4621 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     4681 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     4741 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     4801 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     4861 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     4921 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     4981 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     5041 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     5101 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     5161 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     5221 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     5281 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     5341 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     5401 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     5461 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     5521 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5581 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5641 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5701 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5761 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5821 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5881 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     5941 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6001 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6061 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6121 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6181 gttcctggcc ttttgctggc cttttgctca
//
