LOCUS       sequence-471568-        5530 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of bovine arrestin-1 R175E with inserted GGT 
            codon after starting ATG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5530)
  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 5530)
  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: "Curr 
            Protoc."; date: "2023-09"; volume: "3(9):e832. doi"; pages: " 
            10.1002/cpz1.832"
COMMENT     Sequence Label: BtARR1 R175E
FEATURES             Location/Qualifiers
     source          1..5530
                     /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(1714..1731)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1884..1970
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2062..2089
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2108..2199
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2200..2268,2269..3060)
                     /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(2418..2437)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3231..3819
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3720..3739
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3973..3990
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4005..4145
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4071..4090)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4231..4253
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4331..4408
                     /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             4409..5491
                     /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(4428..4447)
                     /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 ccatgggtaa
      421 ggccaataag ccggcgccaa accacgtgat ctttaaaaag atctcccgtg ataaatcggt
      481 gaccatctac ctggggaaga gagattacat agaccacgtt gaacgagtag agcctgtgga
      541 tggcgtcgtg ctggtggatc ctgagctcgt gaagggcaag agagtgtacg tgtctctgac
      601 gtgtgccttc cgctacggcc aggaagacat cgacgtgatg ggcctcagct tccgcaggga
      661 cctctacttc tcccaggtcc aggtgttccc tcccgtgggg gcctcgggcg ccaccacgag
      721 gctgcaggag agcctcatca agaagctggg ggccaacacc taccccttcc tgctcacgtt
      781 tcctgactac ttgccctgtt cggtgatgct gcagccagct ccgcaagatg tgggcaagag
      841 ctgtggggtc gactttgaga tcaaagcatt cgccacgcac agcacagatg tggaagagga
      901 caaaattccc aagaagagct ccgtgcgtct gctgatcgag aaggtacagc atgcgccgcg
      961 ggatatgggt ccccagcccc gagccgaggc ctcctggcag ttcttcatgt cggacaagcc
     1021 cctgcgcctc gccgtctcgc tcagcaaaga gatctattac cacggggaac ccattcctgt
     1081 gaccgtggcc gtgaccaaca gcacagagaa gacagtgaag aagattaaag tgctagtgga
     1141 gcaagtgacc aacgtggttc tctactcgag tgattattac atcaagacgg tggctgcgga
     1201 ggaagcacag gaaaaagtgc cgccaaacag ctcgctgacc aagacgctga cgctggtgcc
     1261 cttgctggcc aacaaccgtg agagaagggg catcgccctg gatgggaaaa tcaagcacga
     1321 ggacacgaac ctggcctcca gcaccatcat aaaggaggga atagacaaga ccgtcatggg
     1381 gatcctggtg tcttaccaga tcaaggtgaa gctcacggtg tcaggccttc tgggagagct
     1441 cacatccagt gaagtggcca ctgaggtgcc gttccgcctc atgcatcccc agccagagga
     1501 cccagatacc gccaaggaaa gttttcagga tgaaaatttt gtttttgagg agtttgctcg
     1561 ccaaaatctg aaagatgcag gagaatataa ggaagagaag acagaccagg aggcggctat
     1621 ggatgagtga agaggcggct caggacgtgg gatgcggggt cgctagcgcg gccgcaagct
     1681 tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag
     1741 aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc
     1801 catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc
     1861 gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     1921 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag
     1981 cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa
     2041 ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac
     2101 aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2161 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2221 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2281 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2341 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2401 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     2461 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     2521 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     2581 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     2641 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     2701 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     2761 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     2821 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     2881 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     2941 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3001 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3061 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3121 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3181 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3241 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3301 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3361 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     3421 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3481 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3541 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3601 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3661 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3721 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3781 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3841 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     3901 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     3961 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     4021 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
     4081 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     4141 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     4201 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     4261 cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg aagcggcatg
     4321 catttacgtt gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc
     4381 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     4441 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     4501 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     4561 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     4621 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     4681 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     4741 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     4801 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     4861 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     4921 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     4981 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     5041 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     5101 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     5161 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     5221 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     5281 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     5341 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     5401 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     5461 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagcgcg
     5521 aattgatctg
//
