LOCUS       sequence-420221-        6414 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of AqpZ with ALFA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6414)
  AUTHORS   Stover L, Bahramimoghaddam H, Wang L, Schrecke S, Yadav GP, Zhou M, 
            Laganowsky A
  TITLE     Grafting the ALFA tag for structural studies of aquaporin Z.
  JOURNAL   J Struct Biol X. 2024 Feb 2;9:100097. doi: 
            10.1016/j.yjsbx.2024.100097. eCollection 2024 Jun.
  PUBMED    38361954
REFERENCE   2  (bases 1 to 6414)
  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: "J Struct 
            Biol X."; date: "2024-02-2"; volume: "9:100097. doi"; pages: " 
            10.1016/j.yjsbx.2024.100097. eCollection 2024 Jun"
COMMENT     Sequence Label: pET-AqpZ-ALFA
FEATURES             Location/Qualifiers
     source          1..6414
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             522..544
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    559..583
                     /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)."
     primer_bind     complement(583..602)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(584..602)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(686..705)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        911..988
                     /gene="lacI"
                     /label=lacI promoter
     CDS             989..2071
                     /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     complement(1008..1027)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3383..3574
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3568..3590)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3676..3815
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3731..3750
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3830..3847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4001..4589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4081..4100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4760..5551,5552..5620))
                     /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     5383..5402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5621..5725)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5793..5811
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(5838..5843,5844..5860,5861..5866)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(6041..6088)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6084..6102
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(6155..6178)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(6203..6241)
                     /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"
ORIGIN
        1 gggttaaggc cagaccaata gcgatcggcg caaaacctgc cggcgcgaat ttgtcggttg
       61 cgccgtggat caccaacagg aaacctgcac tcaataccag ttcaactacc agcgcggaaa
      121 gcatggaata accgcctggt gaatgctcgc cataaccgtt agaagcaaaa ccgctggctg
      181 ccgcgtcaaa acccgtttta ccactggcaa ttaaatacag cagcgctgcc gcaacaatac
      241 cgccgacaac ctgggcaatt acgtagccaa cgacttcttt tgccggaaaa cgtccgccag
      301 cccataaacc aatagtgacc gccgggttaa aatgaccacc agaaatatga ccaacagcaa
      361 aggccatcgt cagaacggtc agaccgaacg ccaacgccac gccggcaaaa ccaatgccta
      421 attccgggaa gcctgcggcc agtacagcac taccacagcc accaaaaaca agccagaaag
      481 taccaaaaca ttcagctgct aattttctga acatatgtat atctccttct taaagttaaa
      541 caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat
      601 taatttcgcg ggatcgagat ctcgatcctc tacgccggac gcatcgtggc cggcatcacc
      661 ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg
      721 gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg
      781 gccgggggac tgttgggcgc catctccttg catgcaccat tccttgcggc ggcggtgctc
      841 aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt
      901 cgagatcccg gacaccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc
      961 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     1021 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     1081 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     1141 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     1201 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     1261 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     1321 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     1381 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     1441 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     1501 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     1561 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     1621 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     1681 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     1741 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     1801 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     1861 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     1921 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     1981 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     2041 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     2101 ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct
     2161 tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc
     2221 aactcgtagg acaggtgccg gcagcgctct gggtcatttt cggcgaggac cgctttcgct
     2281 ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc
     2341 aagccttcgt cactggtccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg
     2401 gcatggcggc cgacgcgctg ggctacgtct tgctggcgtt cgcgacgcga ggctggatgg
     2461 ccttccccat tatgattctt ctcgcttccg gcggcatcgg gatgcccgcg ttgcaggcca
     2521 tgctgtccag gcaggtagat gacgaccatc agggacagct tcaaggatcg ctcgcggctc
     2581 ttaccagcct aacttcgatc attggaccgc tgatcgtcac ggcgatttat gccgcctcgg
     2641 cgagcacatg gaacgggttg gcatggattg taggcgccgc cctatacctt gtctgcctcc
     2701 ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac ctgaatggaa gccggcggca
     2761 cctcgctaac ggattcacca ctccaagaat tggagccaat caattcttgc ggagaactgt
     2821 gaatgcgcaa accaaccctt ggcagaacat atccatcgcg tccgccatct ccagcagccg
     2881 cacgcggcgc atctcgggca gcgttgggtc ctggccacgg gtgcgcatga tcgtgctcct
     2941 gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc
     3001 gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac
     3061 atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg
     3121 caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac
     3181 atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat
     3241 ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt
     3301 aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa
     3361 tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg
     3421 ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga
     3481 acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct
     3541 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     3601 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     3661 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     3721 cttaactatg cggcatcaga gcagattgta ctgagagtgc accattgcgg tgtgaaatac
     3781 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg
     3841 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3901 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3961 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4021 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4081 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4141 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4201 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4261 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4321 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4381 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4441 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4501 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4561 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4621 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4681 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4741 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4801 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4861 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4921 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4981 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5041 cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt
     5101 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5161 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5221 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5281 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5341 gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata
     5401 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5461 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5521 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5581 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5641 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5701 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     5761 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     5821 ttcaagaatt ctcatgtttg acagcttatc atcgataagc tttaatgcgg tagtttatca
     5881 cagttaaatt gctaacgcag tcaggcaccg tgtatgaaat ctaacaatgc gctcatcgtc
     5941 atcctcggca ccgtcaccct ggatgctgta ggcataggct tggttatgcc ggtactgccg
     6001 ggcctcttgc gggatatccg gatatagttc ctcctttcag caaaaaaccc ctcaagaccc
     6061 gtttagaggc cccaaggggt tatgctagtt attgctcagc ggtggcagca gccaactcag
     6121 cttcctttcg ggctttgtta gcagccggat ctcatttttc aaactgcgga tggctccagc
     6181 tagcgccagg accgccacct ggctctgtaa gacggcggcg aagttcttcc tccaggcgac
     6241 tagcacgcag cagggtccgg taaatcagac caccgataat gccgccgaca attggcacca
     6301 cccagaagaa ccacagttgt tctaatgccc agccgccctg gaagatagca accgcggtgc
     6361 tgcgcgccgg gttaacagaa gtgttagtca ccggaatact aattaagtga atca
//
