LOCUS       sequence-472926-        5606 bp    DNA     circular SYN 15-MAY-2025
DEFINITION  Bacterial expression of bovine arrestin-2 C59V, C125S, C140L, C150V,
            C242V, C251V, C269S.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5606)
  AUTHORS   Zhuo Y, Vishnivetskiy SA, Zhan X, Gurevich VV, Klug CS
  TITLE     Identification of receptor binding-induced conformational changes in
            non-visual arrestins.
  JOURNAL   J Biol Chem. 2014 Jul 25;289(30):20991-1002. doi: 
            10.1074/jbc.M114.560680. Epub 2014 May 27.
  PUBMED    24867953
REFERENCE   2  (bases 1 to 5606)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1074/jbc.M114.560680"; journalName: "J Biol Chem"; date: 
            "2014-07-25- 25"; volume: "289"; issue: "30"; pages: "20991-1002"
COMMENT     Sequence Label: BtARR2 Cys-less
FEATURES             Location/Qualifiers
     source          1..5606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(193..198,199..215,216..222)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    230..246
                     /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     235..257
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    263..332
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             383..409
                     /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"
     primer_bind     complement(1790..1807)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1960..2046
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2138..2165
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2184..2275
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2276..2344,2345..3136)
                     /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(2494..2513)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3307..3895
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3796..3815
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4049..4066
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4081..4221
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4147..4166)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4307..4329
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4407..4484
                     /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             4485..5567
                     /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(4504..4523)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
       61 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
      121 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
      181 tgaaatgagc tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga
      241 taacaatttc acacaggaaa cagcgccgct gagaaaaagc gaagcggcac tgctctttaa
      301 caatttatca gacaatctgt gtgggcactc gaccggaatt atcgattaac tttattatta
      361 aaaattaaag aggtatatat taatgtatcg attaaataag gaggaataaa ccatgggcga
      421 caaagggacg cgggtgttca agaaggcgag ccccaatgga aagctcaccg tctatctggg
      481 aaagcgggac tttgtggacc acatcgacct cgtggagccc gtggatggag tggttcttgt
      541 ggatccggag tatctcaagg agaggagagt ctatgtgacg ctcacggtcg ccttccgcta
      601 cggacgggag gacctggacg tcctgggcct gacctttcgc aaggacctgt ttgtggccaa
      661 cgtgcagtct ttcccgccgg cccctgagga caagaagccc ctgacgcgtc tgcaggagcg
      721 cctcatcaag aagctgggcg agcatgccta ccctttcacc tttgagatcc ctccgaacct
      781 gccaagctct gtgactttgc agccgggacc tgaagataca gggaaggctt taggtgtcga
      841 ctacgaagtg aaagccttcg ttgcggagaa cctggaggag aagatccaca agcggaactc
      901 cgtacgcctg gtcatccgga aggttcagta tgccccagag aggcccgggc cccagcccac
      961 ggccgagacc accaggcagt tcctcatgtc agacaagccc ttgcatctgg aggcctccct
     1021 ggacaaggag atctactacc acggagaacc catcagtgtc aacgtccatg tcaccaacaa
     1081 caccaacaag acggtgaaga agatcaagat ctcggtgcgc cagtatgcag acatcgttct
     1141 gttcaacaca gcccagtaca aggtccctgt ggccatggaa gaggctgatg acacagtggc
     1201 acccagctct acgttcagca aggtctacac gctgaccccc ttcctggcca acaatcgaga
     1261 gaagcggggc ctcgctctag acgggaagct caaacacgag gacacgaacc tggcctcgag
     1321 caccctgttg agggaaggag ccaaccggga aatcctgggc atcattgttt cctacaaagt
     1381 gaaagtgaag ctggtggtgt ctcgtggcgg cctgttggga gatcttgcat ccagtgatgt
     1441 ggccgtggaa ctgcctttca ccctaatgca ccccaagccc aaagaggaac ccccacaccg
     1501 ggaagttcca gagcacgaga cgccggtaga taccaatctc atagaacttg acaccaacga
     1561 tgacgacatt gtgttcgaag actttgcccg ccagagacta aaaggcatga aggatgacaa
     1621 ggaggaagag gaggatggta ccggctctcc gcggctcaac gacagataga ctggggctgc
     1681 cctccctccg ggcagctcca ggtccactct catgcactag gatgcttgtt cgtcttcttc
     1741 ctgtcctggc taagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga
     1801 ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg
     1861 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg
     1921 tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
     1981 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     2041 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     2101 ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc
     2161 ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc
     2221 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2281 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2341 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2401 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2461 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     2521 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2581 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2641 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2701 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2761 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     2821 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2881 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2941 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3001 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3061 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3121 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3181 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3241 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3301 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3361 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3421 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3481 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3541 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3601 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3661 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3721 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3781 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3841 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3901 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3961 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4021 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4081 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
     4141 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     4201 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4261 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4321 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg
     4381 aaggcgaagc ggcatgcatt tacgttgaca ccatcgaatg gtgcaaaacc tttcgcggta
     4441 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     4501 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     4561 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     4621 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     4681 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     4741 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     4801 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     4861 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     4921 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     4981 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     5041 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     5101 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     5161 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     5221 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     5281 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     5341 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     5401 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     5461 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     5521 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     5581 aatgtaagtt agcgcgaatt gatctg
//
