LOCUS       sequence-320675-        6455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the protein expression of BbZIP with ALFA tag at C-terminus in 
            E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6455)
  AUTHORS   Jin F, Shen C, Wang Y, Wang M, Sun M, Hattori M
  TITLE     Fluorescence-detection size-exclusion chromatography utilizing 
            nanobody technology for expression screening of membrane proteins.
  JOURNAL   Commun Biol. 2021 Mar 19;4(1):366. doi: 10.1038/s42003-021-01891-y.
  PUBMED    33742097
REFERENCE   2  (bases 1 to 6455)
  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: "Commun 
            Biol."; date: "2021-03-19"; volume: "4(1):366. doi"; pages: " 
            10.1038/s42003-021-01891-y"
COMMENT     Sequence Label: BbZIP-cALFA
FEATURES             Location/Qualifiers
     source          1..6455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     109..128
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     212..231
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        212..230
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    231..255
                     /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             270..292
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1242..1265
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1275..1313
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             1323..1346
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1359..1391
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             1398..1415
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1468..1486)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1482..1529
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1566..2021
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1653..1672)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1863..1884
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2047..2151
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2152..2220,2221..3012)
                     /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(2370..2389)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3183..3771
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3672..3691
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3925..3942
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3957..4096
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4022..4041)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4182..4204
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4198..4389)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5198..6280)
                     /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
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSY
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6242..6261
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6281..6358)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
       61 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
      121 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
      181 gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
      241 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata
      301 tgaatcagcc gagctccctg gcggcggatc tgcgcggcgc gtggcatgcc caagcgcagt
      361 cacatccgct aattaccttg ggcttagcgg cgagcgcagc gggcgtggtg ctgcttctcg
      421 ttgcgggcat tgtgaacgcg ctgaccggcg aaaaccgcgt ccatgtgggg tatgcggtac
      481 tgggcggcgc ggcgggtttt gctgccactg ctctaggcgc gctaatggcc ctgggactcc
      541 gcgcgattag cgcgcgcacc caagatgcga tgctgggctt cgcggcgggc atgatgctgg
      601 cggcgagcgc gtttagtctg attcttcctg gcttggatgc tgctggaacg atagtaggtc
      661 ctggaccggc cgctgcggcg gtagtggcgc tcggcctggg actaggcgtg ctgctgatgc
      721 tgggcctgga ttattttacc ccgcatgaac atgaacgcac cggccatcaa ggcccggaag
      781 cggcgcgcgt gaaccgcgtg tggctgtttg tgctgaccat tattctgcat aacctgccgg
      841 aaggcatggc gattggcgtg agctttgcga ccggcgatct gcgcattggc ctgccgctga
      901 cgagcgcgat tgcaattcaa gatgtaccgg aaggcttagc ggttgcactg gcgctgcgcg
      961 ccgtggggtt acctattggt cgcgcggtac tggtcgccgt cgcgagcggc ctaatggaac
     1021 ccctgggcgc gcttgtggga gtgggcatta gcagcggctt tgcgctggcg tatccgatta
     1081 gcatgggatt agctgcggga gcaatgattt ttgtggtgag ccatgaagtg attccggaaa
     1141 cccatcgcaa cggccatgaa accaccgcga ccgtgggcct gatggctggg tttgcgctga
     1201 tgatgtttct ggataccgct ctgggcctcg agagtagtgg cctggaagtt ctgttccagg
     1261 ggccctttac tccgagccgt ctggaagaag aactgcgccg ccgtctgacc gaaccagcca
     1321 gtcaccacca ccaccaccac caccactaat gagctagcca gccagaactc gccccggaag
     1381 accccgagga tgtcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     1441 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1501 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     1561 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1621 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1681 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1741 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1801 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     1861 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     1921 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     1981 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     2041 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2101 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     2161 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     2221 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     2281 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     2341 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     2401 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     2461 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2521 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     2581 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2641 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     2701 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     2761 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     2821 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     2881 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     2941 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3001 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     3061 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     3121 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3181 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3241 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3301 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     3361 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     3421 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3481 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3541 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3601 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3661 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3721 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3781 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3841 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3901 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     3961 atgcggtatt ttctccttgc gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     4021 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     4081 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4141 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4201 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     4261 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     4321 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     4381 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     4441 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     4501 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     4561 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     4621 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     4681 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     4741 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     4801 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     4861 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     4921 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     4981 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     5041 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     5101 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     5161 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5221 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5281 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     5341 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     5401 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     5461 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     5521 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     5581 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     5641 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     5701 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     5761 gatgctccac gcccagtcgc gtaccgtctt cataggagaa aataatactg ttgatgggtg
     5821 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     5881 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     5941 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     6001 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     6061 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     6121 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     6181 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     6241 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     6301 tctcttctgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     6361 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     6421 aggccgttga gcaccgccgc cgcaaggaat ggtgc
//
