LOCUS       sequence-471664-        5670 bp    DNA     circular SYN 06-MAY-2025
DEFINITION  Bacterial expression of bovine arrestin-2 long isoform (1-382) 
            D383Stop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5670)
  AUTHORS   Vishnivetskiy SA, Zhan X, Gurevich VV
  TITLE     Expression of Untagged Arrestins in E. coli and Their Purification.
  JOURNAL   Curr Protoc. 2023 Sep;3(9):e832. doi: 10.1002/cpz1.832.
  PUBMED    37671938
REFERENCE   2  (bases 1 to 5670)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Curr 
            Protoc."; date: "2023-09"; volume: "3(9):e832. doi"; pages: " 
            10.1002/cpz1.832"
COMMENT     Sequence Label: BtARR2 long (1-382)
FEATURES             Location/Qualifiers
     source          1..5670
                     /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     1796..1812
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1797..1813
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1854..1871)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2024..2110
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2202..2229
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2248..2339
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2340..2408,2409..3200)
                     /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(2558..2577)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3371..3959
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3860..3879
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4113..4130
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4145..4285
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4211..4230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4371..4393
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4471..4548
                     /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             4549..5631
                     /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(4568..4587)
                     /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 ctgacctgcg ccttccgcta
      601 cggccgggag gacctggatg tcctgggcct gacctttcgc aaggacctgt ttgtggccaa
      661 cgtgcagtct ttcccgccgg cccctgagga caagaagccc ctgacgcggc tgcaggagcg
      721 cctcatcaag aagctgggcg agcatgccta ccctttcacc tttgagatcc ctccgaacct
      781 cccatgctct gtgactttgc agccgggacc tgaagataca gggaaggcct gcggtgtgga
      841 ctacgaagtg aaagccttct gtgcggagaa cctggaggag aagatccaca agcggaattc
      901 tgtgcgcctg gtcatccgga aggttcagta tgccccagag aggcctggcc cccagcccac
      961 ggccgagacc accaggcagt tcctcatgtc agacaagccc ttgcatctgg aggcctccct
     1021 ggacaaggag atctactacc acggagaacc catcagtgtc aacgtccatg tcaccaacaa
     1081 caccaacaag acggtgaaga agatcaagat ctcggtgcgc cagtatgcag acatctgtct
     1141 gttcaacaca gcccagtaca agtgccctgt ggccatggaa gaggctgatg acacagtggc
     1201 acccagctct acgttctgca aggtctacac gctgaccccc ttcctggcca acaatcgaga
     1261 gaagcggggc ctcgccctgg acgggaagct caaacacgag gacacgaacc tggcctccag
     1321 caccctgttg agggaaggag ccaaccggga gatcctgggc atcattgttt cctacaaagt
     1381 gaaagtgaag ctggtggtgt ctcgtggcgg cctgttggga gatcttgcat ccagtgatgt
     1441 ggccgtggaa ctgcctttca ccctaatgca ccccaagccc aaagaggaac ccccacaccg
     1501 ggaagttcca gagcacgaga cgccggtaga taccaatctc atagaacttg acaccaacta
     1561 ggacgacatt gtgtttgagg actttgcccg ccagagacta aaaggcatga aggatgacaa
     1621 ggaggaagag gaggatggta ccggctctcc gcggctcaac gacagataga ctggggctgc
     1681 cctccctccg ggcagctcca ggtccactct catgcactag gatgcttgtt cgtcttcttc
     1741 ctgtcctggc tccccctccc ctttgttctt ccagtttcta ccagggggcc ccccctcgag
     1801 gtcgacggta tcgataagct tggctgtttt ggcggatgag agaagatttt cagcctgata
     1861 cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc
     1921 gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt
     1981 agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc
     2041 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     2101 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg
     2161 ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga
     2221 tggccttttt gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt
     2281 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     2341 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     2401 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     2461 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     2521 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     2581 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     2641 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     2701 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     2761 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2821 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2881 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2941 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     3001 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     3061 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3121 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3181 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     3241 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3301 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3361 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3421 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3481 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     3541 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3601 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3661 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3721 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3781 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3841 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3901 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3961 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4021 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4081 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4141 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     4201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     4261 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     4321 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4381 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg
     4441 cgcgaaggcg aagcggcatg catttacgtt gacaccatcg aatggtgcaa aacctttcgc
     4501 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta
     4561 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     4621 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     4681 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     4741 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     4801 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     4861 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     4921 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     4981 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa
     5041 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     5101 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat
     5161 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc
     5221 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
     5281 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt
     5341 ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg
     5401 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
     5461 ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg
     5521 aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     5581 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     5641 aattaatgta agttagcgcg aattgatctg
//
