LOCUS       sequence-369594-        5955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector containing N-terminal 6His-GB1-TEV 
            fusion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5955)
  AUTHORS   Venkatesan M, Semper C, Skrivergaard S, Di Leo R, Mesa N, Rasmussen 
            MK, Young JF, Therkildsen M, Stogios PJ, Savchenko A
  TITLE     Recombinant production of growth factors for application in cell 
            culture.
  JOURNAL   iScience. 2022 Sep 3;25(10):105054. doi: 10.1016/j.isci.2022.105054.
            eCollection 2022 Oct 21.
  PUBMED    36157583
REFERENCE   2  (bases 1 to 5955)
  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: "iScience.";
            date: "2022-09-3"; volume: "25(10):105054. doi"; pages: " 
            10.1016/j.isci.2022.105054. eCollection 2022 Oct 21"
COMMENT     Sequence Label: pMCSGC-GB1
FEATURES             Location/Qualifiers
     source          1..5955
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(99..118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        285..388
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(389..457,458..1249)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(607..626)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1420..2008
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1909..1928
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2162..2179
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2259..2278)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(2434..3516)
                     /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     3478..3497
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3517..3594)
                     /gene="lacI"
                     /label=lacI promoter
     tRNA            3734..3809
                     /gene="ileX"
                     /product="E. coli tRNA-Ile recognizing AUA"
                     /label=ileX
     primer_bind     3958..3977
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     tRNA            4079..4155
                     /gene="argU"
                     /product="E. coli tRNA-Arg recognizing AGA and AGG"
                     /label=argU
     primer_bind     4430..4449
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4430..4448
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4449..4473
                     /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             4488..4510
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4521..4538
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4563..4721
                     /codon_start=1
                     /product="B1 domain of Streptococcal protein G   "
                     /label=GB1
                     /note="effective as a solubilizing fusion partner (Cheng 
                     and Patel, 2004)"
                     /translation="KLALNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDA
                     TKTFTVTE"
     CDS             4728..4748
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             complement(4994..5299)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(5096..5115)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             5799..5816
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5869..5887)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5883..5930
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
      301 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
      361 tgcttcaata atttgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      421 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      481 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      541 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      601 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
      661 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      721 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      781 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      841 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      901 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
      961 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1021 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1081 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1141 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1201 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1261 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1321 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1381 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1441 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1501 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1561 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1621 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1681 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1741 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1801 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1861 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1921 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1981 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2041 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2101 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2161 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2221 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     2281 tagttaagcc agtatacact ccgctatcgc tacgtgactg tagtcatgcc ccgcgcccac
     2341 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     2401 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2461 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2521 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     2581 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     2641 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     2701 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     2761 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     2821 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     2881 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     2941 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     3001 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     3061 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     3121 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     3181 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     3241 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     3301 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     3361 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     3421 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     3481 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     3541 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     3601 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     3661 cgttgagcac cgccgccgca aggaatggtg catgcggatt gcgacacgga gttactttat
     3721 aatccgctac catggcccct tagctcagtg gttagagcag gcgactcata atcgcttggt
     3781 cgctggttca agtccagcag gggccaccag atatagcaaa ggctgacgag aaatcgtcag
     3841 cctttttgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     3901 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     3961 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggcgagat caaaagccat
     4021 tgactcagca agggttgacc gtataattca cgcgattaca ccgcattgcg gtatcaacgc
     4081 gcccttagct cagttggata gagcaacgac cttctaagtc gtgggccgca ggttcgaatc
     4141 ctgcagggcg cgccattaca attcaatcag ttacgccttc tttatatcct ccataattcc
     4201 agagtgggac atatttggga cattatcacc aaaaatgtcg tctattttcc tcgcatgctc
     4261 tgtcaaatga ttaggcgcaa ggtgagcata cctacgaacc atttctatgg actcccatcc
     4321 gcccatttcc tgaagcactg ataatgggac gcctgactga atccaccggt gatgccggcc
     4381 acgatgcgtc cggcgtagag gatcgagatc gatctcgatc ccgcgaaatt aatacgactc
     4441 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     4501 aagaaggaga tatacatatg caccatcatc atcatcattc ttctggtgta gatctgggta
     4561 ccaagcttgc tctgaacggt aaaaccctga aaggtgaaac caccaccgaa gctgttgacg
     4621 ctgctaccgc ggaaaaagtt ttcaaacagt acgctaacga caacggtgtt gacggtgaat
     4681 ggacctacga cgacgctacc aaaaccttca cggtaaccga aggtaccgag aacctgtact
     4741 tccaatccaa tattgcgtgc gagcctcttt tttgtgtgac aaaataaaaa catctaccta
     4801 ttcatatacg ctagtgtcat agtcctgaaa atcatctgca tcaagaacaa tttcacaact
     4861 cttatacttt tctcttacaa gtcgttcggc ttcatctgga ttttcagcct ctatacttac
     4921 taaacgtgat aaagtttctg taatttctac tgtatcgacc tgcagactgg ctgtgtataa
     4981 gggagcctga catttatatt ccccagaaca tcaggttaat ggcgtttttg atgtcatttt
     5041 cgcggtggct gagatcagcc acttcttccc cgataacgga gaccggcaca ctggccatat
     5101 cggtggtcat catgcgccag ctttcatccc cgatatgcac caccgggtaa agttcacggg
     5161 agactttatc tgacagcaga cgtgcactgg ccagggggat caccatccgt cgcccgggcg
     5221 tgtcaataat atcactctgt acatccacaa acagacgata acggctctct cttttatagg
     5281 tgtaaacctt aaactgcatt tcaccagccc ctgttctcgt cagcaaaaga gccgttcatt
     5341 tcaataaacc gggcgacctc agccatccct tcctgatttt ccgctttcca gcgttcggca
     5401 cgcagacgac gggcttcatt ctgcatggtt gtgcttacca gaccggagat attgacatca
     5461 tatatgcctt gagcaactga tagctgtcgc tgtcaactgt cactgtaata cgctgcttca
     5521 tagcatacct ctttttgaca tacttcgggt atacatatca gtatatattc ttataccgca
     5581 aaaatcagcg cgcaaatacg catactgtta tctggctttt agtaagccgg atccacgcgg
     5641 cgtttacgcc ccccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg
     5701 acatggaagc catcacagac ggcatgatga accaatattg gaagtggata acggatccga
     5761 attcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac caccactgag
     5821 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     5881 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     5941 gaactatatc cggat
//
