LOCUS       sequence-243787-        6862 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli. exp. and purif. of X. laevis cytATL-R232Q fragment fused to
            GFP in c-terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6862)
  AUTHORS   Wang S, Tukachinsky H, Romano FB, Rapoport TA
  TITLE     Cooperation of the ER-shaping proteins atlastin, lunapark, and 
            reticulons to generate a tubular membrane network.
  JOURNAL   Elife. 2016 Sep 13;5. pii: e18605. doi: 10.7554/eLife.18605.
  PUBMED    27619977
REFERENCE   2  (bases 1 to 6862)
  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: "Elife."; 
            date: "2016-09-13"; volume: "5. pii: e18605. doi"; pages: " 
            10.7554/eLife.18605"
COMMENT     Sequence Label: pProEx-Htb-cytATL2(R323Q)-6Gly-sfGFP
FEATURES             Location/Qualifiers
     source          1..6862
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(837..1265)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(947..968)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1159..1178
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(1303..1322)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1495..1517
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        1595..1672
                     /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."
     CDS             1673..2755
                     /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(1692..1711)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    3024..3040
                     /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     3029..3051
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             3073..3090
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3112..3132
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4546..5259
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             4546..5256
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(4573..4601)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5196..5216
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(5346..5363)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      5516..5602
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5694..5721
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        5740..5831
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5832..5900,5901..6692)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6050..6069)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcataattt tgttaaaatt
      841 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
      901 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
      961 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     1021 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     1081 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     1141 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     1201 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     1261 cgcgtcccat tcgccattca ggctgctatg gtgcactctc agtacaatct gctctgatgc
     1321 cgcatagtta agccagtacc agtcacgtag cgatatcgct acgtgactgg tacactccgc
     1381 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     1441 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     1501 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag cagatcaatt
     1561 cgcgcgcgaa ggcgaagcgg catgcattta cgttgacacc atcgaatggt gcaaaacctt
     1621 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     1681 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     1741 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     1801 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     1861 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     1921 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     1981 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     2041 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     2101 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     2161 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     2221 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     2281 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     2341 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     2401 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     2461 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     2521 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     2581 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     2641 ggtgaaaaga aaaaccaccc tggcacccaa tacgcaaacc gcctctcccc gcgcgttggc
     2701 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2761 acgcaattaa tgtaagttag cgcgaattga tctggtttga cagcttatca tcgactgcac
     2821 ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt
     2881 cgtaaatcac tgcataattc gtgtcgctca aggcgcactc ccgttctgga taatgttttt
     2941 tgcgccgaca tcataacggt tctggcaaat attctgaaat gagctgttga caattaatca
     3001 tccggtccgt ataatctgtg gaattgtgag cggataacaa tttcacacag gaaacagacc
     3061 atgtcgtact accatcacca tcaccatcac gattacgata tcccaacgac cgaaaacctg
     3121 tattttcagg gcgccatggg atccgccgaa ggcggtagcc tgcgtaaccg cacccgtttc
     3181 ggcagtcgca gcaacgaagc aatgaatcat gttgattatc cggatgaaaa ctttgtggaa
     3241 gaaattcagc tgaatagtga taccgaagtt atggaaaaac cgcgcccgat tcagatcgtg
     3301 ctggcacatg aagatgatca caacttcgaa ctggatgaaa cggcgctgga aagcattctg
     3361 atgcaggatc acatccgtga tctgaatgtg gttgtgctga gcgtggccgg tgcatttcgc
     3421 aaaggcaaat cttttctgct ggatttcatg ctgcgtttta tgtacagcca gagcagcagc
     3481 tggattggcg gtaattatga accgctgacc ggtttcacgt ggcgcggcgg ttgcgaacgt
     3541 gaaaccacgg gcattcagat ctggagtgaa gtttttgttg tggaaaaacc ggatggtagc
     3601 aaagttgcgg tgattctgat ggatacccag ggcgccttcg attctcagag taccatcaaa
     3661 gattgtgcaa cggtgtttgc gctgtctacc atgaccagca gcgttcaggt gtacaacctg
     3721 agtcagaata ttcaggaaga tgatctgcag catctgcagc tgttcaccga atacggtcgc
     3781 ctggcgatgg aagaaattta taaaaaaccg ttccagacgc tgatgtttct gatccaggat
     3841 tggtcttatc cgtacgaaca tagttatggc ctggaaggcg gtaacaaatt tctggaaaaa
     3901 cgcctgcagg ttaaacagaa ccagcacgaa gaactgcaga atgtgcgtaa acatattcac
     3961 tcttgcttca ccaacatcgg ttgttttctg ctgccgcatc cgggcctgaa agttgccacg
     4021 aatccgtatt tcgatggtcg cctggtggaa atcgatgatg aatttaaaaa agaactgcgt
     4081 gaactggttc cgctgctgct ggcaccggaa aatctggttg aaaaagaaat cagcggttct
     4141 aaagtgacct gccgcgatct gctggaatac ttcaaagcgt acatcaaaat ctaccagggc
     4201 gaagaactgc cgcacccgaa aagcatgctg caggcgaccg ccgaagcaaa caatatggcg
     4261 gccgttgcgg gcgccaaaga tacgtacagt cgtagcatgg aacaagtgtg tggcggtgat
     4321 aaaccgtata ttgcgccgag cgatctggaa cgcaaacatc tggatctgaa agaaacctgc
     4381 atcaaacagt tccgttctgt gaagaaaatg ggcggtgaag aattttgtcg tcgctaccag
     4441 gaacagctgg aagccgaaat tgaagaaacc tatgcaaact tcctgaaaca caacgatggt
     4501 aaaaacatct tttacgcagc acgtaccggc ggcggcggcg gcggcagcaa aggagaagaa
     4561 cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa
     4621 ttttctgtcc gtggagaggg tgaaggtgat gctacaaacg gaaaactcac ccttaaattt
     4681 atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac tttaacttat
     4741 ggtgttcaat gcttttcccg ttatccggat catatgaaac ggcatgactt tttcaagagt
     4801 gccatgcccg aaggttatgt acaggaacgc actatatctt tcaaagatga cgggacctac
     4861 aagacgcgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatcgtat cgagttaaaa
     4921 ggtattgatt ttaaagaaga tggaaacatt ctcggacaca aactcgagta caactttaac
     4981 tcacacaatg tatacatcac ggcagacaaa caaaagaatg gaatcaaagc taacttcaaa
     5041 attcgccaca acgttgaaga tggttccgtt caactagcag accattatca acaaaatact
     5101 ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtcgac acaatctgtc
     5161 ctttcgaaag atcccaacga aaagcgtgac cacatggtcc ttcttgagtt tgtaactgct
     5221 gctgggatta cacatggcat ggatgagctc tacaaataag cggccgcttt cgaatctaga
     5281 gcctgcagtc tcgaggcatg cggtaccaag cttggctgtt ttggcggatg agagaagatt
     5341 ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct
     5401 ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt
     5461 agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat
     5521 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     5581 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
     5641 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc
     5701 catcctgacg gatggccttt ttgcgtttct acaaactctt ttgtttattt ttctaaatac
     5761 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5821 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5881 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5941 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     6001 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     6061 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     6121 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     6181 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     6241 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6301 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6361 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6421 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6481 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6541 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6601 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6661 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6721 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6781 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6841 tagaaaagat caaaggatct tc
//
