LOCUS       sequence-52461-e        6504 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 6504)
  AUTHORS   Silvers R, Saxena K, Kudlinzki D, Schwalbe H
  TITLE     Recombinant expression and purification of human TATA binding 
            protein using a chimeric fusion.
  JOURNAL   Protein Expr Purif. 2012 Jul 24;85(1):142-147.
  PUBMED    22841618
REFERENCE   2  (bases 1 to 6504)
  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"; journalName: "Protein 
            Expr Purif."; date: "2012-07-24"; volume: "85(1)"; pages: "142-147"
COMMENT     Sequence Label: pRS-45
FEATURES             Location/Qualifiers
     source          1..6504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             14..31
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             41..58
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             293..313
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(916..934)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      930..977
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1152..1157,1158..1174,1175..1180))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1207..1225)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1293..1397
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1398..1466,1467..2258)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1616..1635)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2429..3017
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2918..2937
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3171..3188
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3203..3345
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3271..3290)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3431..3453
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3447..3638)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4950..6032)
                     /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     5994..6013
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6033..6110)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6316..6335
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6419..6438
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6419..6437
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6438..6462
                     /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             6477..6499
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 catgggcagc tctcatcacc atcaccatca cagtagcggt ctggtgccgc gtggtagcca
       61 catgggcaaa accaacctgg ccaatgaaga tgaagcgtat gaagccattt ttggcggtga
      121 attcggttct ctggaaattg gcagttatat cggcggtgat gaaggcgcga actctaaaga
      181 ttacacggaa cacctgccgg atgcagttga ttttgaagat gaagatgaac tggcggatga
      241 tgatgatgat ctgccgggcg gtggctctgg tggcggtagt ggcggtggca gcgagaatct
      301 ttattttcag ggccatatga gcggcattgt tccgcagctg cagaatattg tgagcaccgt
      361 taatctgggt tgcaaactgg atctgaaaac cattgccctg cgcgcccgca acgcggaata
      421 taacccgaaa cgcttcgcgg cggttattat gcgcattcgc gaaccgcgta ccaccgcgct
      481 gatttttagc agcggtaaaa tggtgtgcac cggcgccaaa agcgaagaac agagccgtct
      541 ggcggcccgt aaatatgcgc gcgttgttca gaaactgggc tttccggcga aatttctgga
      601 ttttaaaatt cagaacatgg tgggcagctg cgatgttaaa tttccgattc gtctggaagg
      661 cctggttctg acccatcagc agtttagcag ctatgaaccg gaactgtttc cgggtctgat
      721 ttatcgtatg atcaaaccgc gtattgtgct gctgattttt gtgagcggca aagttgtgct
      781 gaccggtgcg aaagttcgtg cggaaattta tgaagcgttt gaaaacattt atccgatcct
      841 gaaaggtttt cgcaaaacca cctaaggatc cggctgctaa caaagcccga aaggaagctg
      901 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
      961 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atatcccgca agaggcccgg
     1021 cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac
     1081 gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga
     1141 taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac
     1201 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     1261 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1321 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1381 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1441 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1501 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1561 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1621 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     1681 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1741 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     1801 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     1861 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     1921 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     1981 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2041 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2101 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2161 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2221 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2281 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2341 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2401 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2461 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2521 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2581 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2641 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2701 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2761 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     2821 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     2881 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     2941 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3001 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3061 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3121 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3181 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3241 tttcacaccg catatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3301 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     3361 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3421 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3481 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     3541 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     3601 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     3661 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     3721 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     3781 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     3841 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     3901 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     3961 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     4021 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     4081 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
     4141 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
     4201 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
     4261 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
     4321 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
     4381 ccgaggcggc ataaatcgcc gtgacgatca gcggtccagt gatcgaagtt aggctggtaa
     4441 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
     4501 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
     4561 ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc
     4621 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     4681 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     4741 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     4801 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     4861 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     4921 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4981 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5041 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     5101 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     5161 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     5221 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     5281 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     5341 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     5401 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5461 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5521 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5581 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     5641 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     5701 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     5761 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     5821 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     5881 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     5941 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     6001 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     6061 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     6121 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     6181 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     6241 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     6301 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     6361 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
     6421 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     6481 tttaacttta agaaggagat atac
//
