LOCUS       sequence-382970-        4999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of SARS-CoV2 receptor binding domain in E. coli with 
            C-terminal Avi-tag .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4999)
  AUTHORS   Fischer K, Lulla A, So TY, Pereyra-Gerber P, Raybould MIJ, Kohler 
            TN, Kaminski TS, Yam-Puc JC, Hughes R, Leiß-Maier F, Brear P, 
            Matheson NJ, Deane CM, Hyvonen M, Thaventhiran JED, Hollfelder F
  TITLE     Microfluidics-enabled fluorescence-activated cell sorting of single 
            pathogen-specific antibody secreting cells for the rapid discovery 
            of monoclonal antibodies
  JOURNAL   bioRxiv 2023.01.10.523494
REFERENCE   2  (bases 1 to 4999)
  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: "bioRxiv 
            2023.01.10.523494"
COMMENT     Sequence Label: pExp-RBD-CHis
FEATURES             Location/Qualifiers
     source          1..4999
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..28
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        9..27
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    27..51
                     /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             64..86
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             94..120
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding 
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /label=minicistron
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             724..747
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     promoter        complement(761..779)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(762..779)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(837..855)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      851..898
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1134..1155)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1305..2387)
                     /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     2349..2368
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2388..2465)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     2604..2623
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2777..2796)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2896..2918
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2956..2974)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3042..3146
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3147..3215,3216..4007)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3365..3384)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4178..4766
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4667..4686
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4920..4937
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagctgatta atacgactca ctatagggaa ttgtgagcgg ataacaattc ctctagaaat
       61 aattttgttt aactttaaga aggagatata tccatgtatc gattaaataa ggaggaataa
      121 accatgaata tcactaacct gtgcccgttc ggtgaagttt tcaacgcgac ccgtttcgcg
      181 tccgtctacg cgtggaaccg taaacgtatc tctaactgcg ttgcggacta ctctgtcctg
      241 tacaactctg cgtctttctc tacctttaaa tgctacggtg tttctccgac caaactgaac
      301 gacctctgct ttaccaacgt ttatgccgac tctttcgtta tccgtggtga cgaagttcgt
      361 cagatcgcgc caggtcaaac cggcaaaatc gcggattaca attacaaact gccggacgat
      421 ttcaccggtt gtgttattgc ttggaactct aacaacctgg actctaaagt tggtggtaac
      481 tacaactacc tgtatcgtct gttccgtaaa tctaacctca aaccatttga acgtgacatc
      541 agcaccgaaa tctaccaggc gggttctacc ccgtgcaacg gtgtcgaggg tttcaactgc
      601 tacttcccgc tgcagtctta tggtttccag cctactaacg gcgttggtta ccagccgtac
      661 cgtgttgttg ttctgtcttt cgaactgctg cacgctcctg ctaccgtttg tggctcgagc
      721 cctcatcacc atcaccatca ccatcactaa gcttgagtat tctatagtgt cacctaaatc
      781 ccagcttgat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
      841 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
      901 gaaaggagga actatatccg gataacctgg cgtaatagcg aagaggcccg caccgatcgc
      961 ccttcccaac agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt aatattttgt
     1021 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     1081 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     1141 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     1201 atcagggcga tggcccacag cttgcatgcc tgcaggtcgg aagcataaag tgtaaagcct
     1261 ggggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     1321 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1381 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     1441 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     1501 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     1561 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     1621 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     1681 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     1741 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     1801 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     1861 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     1921 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     1981 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     2041 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     2101 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     2161 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     2221 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     2281 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     2341 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     2401 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg
     2461 gtgtcggcga gctcgaattg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     2521 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     2581 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     2641 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     2701 cagggcgcgt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     2761 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     2821 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     2881 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     2941 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     3001 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3061 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3121 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3181 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3241 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3301 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3361 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     3421 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3481 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3541 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3601 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3661 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3721 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3781 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3841 aatctggagc cggtgagcgt ggttctcgcg gtatcattgc agcactgggg ccagatggta
     3901 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     3961 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4021 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4081 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4141 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4201 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4261 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4321 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4381 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4441 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4501 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4561 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4621 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4681 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4741 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4801 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4861 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4921 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     4981 gttggccgat tcattaatg
//
