LOCUS       sequence-488759-        5640 bp    DNA     circular SYN 08-OCT-2025
DEFINITION  Expression of mutated bovine arrestin-2 (I386A, V387A, F388A) in 
            bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5640)
  AUTHORS   Zheng C, Weinstein LD, Nguyen KK, Grewal A, Gurevich EV, Gurevich VV
  TITLE     GPCR Binding and JNK3 Activation by Arrestin-3 Have Different 
            Structural Requirements.
  JOURNAL   Cells. 2023 Jun 6;12(12):1563. doi: 10.3390/cells12121563.
  PUBMED    37371033
REFERENCE   2  (bases 1 to 5640)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 8, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cells."; 
            date: "2023-06-6"; volume: "12(12):1563. doi"; pages: " 
            10.3390/cells12121563"
COMMENT     Sequence Label: BtARR2-3A
FEATURES             Location/Qualifiers
     source          1..5640
                     /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(1824..1841)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1994..2080
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2172..2199
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2218..2309
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2310..2378,2379..3170)
                     /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(2528..2547)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3341..3929
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3830..3849
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4083..4100
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4115..4255
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4181..4200)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4341..4363
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4441..4518
                     /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             4519..5601
                     /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(4538..4557)
                     /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 ctcacgtgcg ccttccgcta
      601 cggacgggag gacctggacg tcctgggcct gacctttcgc aaggacctgt ttgtggccaa
      661 cgtgcagtct ttcccgccgg cccctgagga caagaagccc ctgacgcgtc tgcaggagcg
      721 cctcatcaag aagctgggcg agcatgccta ccctttcacc tttgagatcc ctccgaacct
      781 gccatgctct gtgactttgc agccgggacc tgaagataca gggaaggctt gcggtgtcga
      841 ctacgaagtg aaagccttct gtgcggagaa 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 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 acaccaacga
     1561 tgacgacgct gcggctgagg actttgcccg ccagagacta aaaggcatga aggatgacaa
     1621 ggaggaagag gaggatggta ccggctctcc gcggctcaac gacagataga ctggggctgc
     1681 cctccctccg ggcagctcca ggtccactct catgcactag gatgcttgtt cgtcttcttc
     1741 ctgtcctggc tccccctccc ctttgttctt ccagtttcta ccaggaagct tggctgtttt
     1801 ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg
     1861 ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac
     1921 tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg
     1981 aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
     2041 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa
     2101 cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca
     2161 tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctttt
     2221 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     2281 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     2341 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     2401 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     2461 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     2521 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     2581 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     2641 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     2701 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     2761 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     2821 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     2881 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2941 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3001 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     3061 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3121 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3181 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3241 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3301 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3361 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3421 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3481 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3541 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3601 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3661 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3721 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3781 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3841 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3901 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3961 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4021 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4081 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     4141 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
     4201 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     4261 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4321 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     4381 gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg aagcggcatg catttacgtt
     4441 gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     4501 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     4561 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     4621 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     4681 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     4741 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     4801 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     4861 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     4921 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     4981 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     5041 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     5101 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     5161 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     5221 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     5281 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     5341 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     5401 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     5461 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     5521 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     5581 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagcgcg aattgatctg
//
