LOCUS       sequence-382968-        5239 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 5239)
  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 5239)
  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-His-zBasic-RBD-Avi
FEATURES             Location/Qualifiers
     source          1..5239
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        11..29
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    29..53
                     /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             66..88
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             102..125
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             141..311
                     /codon_start=1
                     /product="Z domain of staphylococcal protein A, engineered 
                     to contain multiple positive charges (Hedhammar and Hober, 
                     2007)"
                     /label=Z-basic tag
                     /note="useful for purifying tagged proteins by 
                     cation-exchange chromatography"
                     /translation="VDNKFNKERRRARREIRHLPNLNREQRRAFIRSLRDDPSQSANLL
                     AEAKKLNDAQPK"
     CDS             321..341
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             942..986
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     promoter        complement(1003..1021)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1004..1021)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1079..1097)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1093..1140
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1376..1397)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1547..2629)
                     /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     2591..2610
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2630..2707)
                     /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     2846..2865
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3019..3038)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3138..3160
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3198..3216)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3284..3388
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3389..3457,3458..4249)
                     /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(3607..3626)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4420..5008
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4909..4928
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5162..5179
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tgcagctgat taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa
       61 ataattttgt ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc
      121 accattctgg cactagtggc gttgacaaca aattcaacaa agaacgtcgc cgtgcgcgtc
      181 gcgaaatccg tcacctgccg aacctcaacc gtgaacagcg tcgtgcgttc atccgttctc
      241 tgcgtgacga cccgtctcag tctgcgaacc tgctggcgga agcgaagaaa ctgaacgacg
      301 cacaaccgaa aagcggtacc gaaaacctgt acttccaggg ttctggtact aacctgtgcc
      361 cgttcggtga agttttcaac gcgacccgtt tcgcgtccgt ctacgcgtgg aaccgtaaac
      421 gtatctctaa ctgcgttgcg gactactctg tcctgtacaa ctctgcgtct ttctctacct
      481 ttaaatgcta cggtgtttct ccgaccaaac tgaacgacct ctgctttacc aacgtttatg
      541 ccgactcttt cgttatccgt ggtgacgaag ttcgtcagat cgcgccaggt caaaccggca
      601 aaatcgcgga ttacaattac aaactgccgg acgatttcac cggttgtgtt attgcttgga
      661 actctaacaa cctggactct aaagttggtg gtaactacaa ctacctgtat cgtctgttcc
      721 gtaaatctaa cctcaaacca tttgaacgtg acatcagcac cgaaatctac caggcgggtt
      781 ctaccccgtg caacggtgtc gagggtttca actgctactt cccgctgcag tcttatggtt
      841 tccagcctac taacggcgtt ggttaccagc cgtaccgtgt tgttgttctg tctttcgaac
      901 tgctgcacgc tcctgctacc gtttgtggtc cgggctcgag cggactgaat gacattttcg
      961 aagcacagaa gatcgaatgg catgaagcct aagcttgagt attctatagt gtcacctaaa
     1021 tcccagcttg atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1081 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1141 ctgaaaggag gaactatatc cggataacct ggcgtaatag cgaagaggcc cgcaccgatc
     1201 gcccttccca acagttgcgc agcctgaatg gcgaatggaa attgtaagcg ttaatatttt
     1261 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     1321 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     1381 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     1441 ctatcagggc gatggcccac agcttgcatg cctgcaggtc ggaagcataa agtgtaaagc
     1501 ctggggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     1561 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     1621 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     1681 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     1741 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     1801 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     1861 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     1921 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     1981 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     2041 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     2101 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     2161 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     2221 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     2281 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     2341 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     2401 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     2461 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     2521 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     2581 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     2641 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcga
     2701 tggtgtcggc gagctcgaat tgtgaaccat caccctaatc aagttttttg gggtcgaggt
     2761 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     2821 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     2881 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     2941 tacagggcgc gtcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3001 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     3061 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     3121 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     3181 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     3241 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3301 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3361 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     3421 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3481 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3541 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3601 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     3661 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     3721 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     3781 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3841 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3901 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     3961 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     4021 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     4081 taaatctgga gccggtgagc gtggttctcg cggtatcatt gcagcactgg ggccagatgg
     4141 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     4201 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     4261 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4321 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4381 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4441 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4501 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4561 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4621 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4681 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4741 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4801 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4861 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4921 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4981 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5041 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5101 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5161 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     5221 gcgttggccg attcattaa
//
