LOCUS       sequence-488534-        6676 bp    DNA     circular SYN 07-OCT-2025
DEFINITION  Expression of mutated bovine arrestin-3 (deleted residues 175, 177, 
            178, 182-185) tagged with SmBiT and Flag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6676)
  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 6676)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 7, 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: pSmBtA3-d7F
FEATURES             Location/Qualifiers
     source          1..6676
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2188..2211
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2229..2247)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2230..2247)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2267..2284)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2273..2497
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2543..2971
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2630..2649)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2840..2861
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2980..3000)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2985..3314
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3165..3300
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3227..3246
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3381..4175
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3435..3454)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4045..4064
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4349..4470
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4386..4405)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4440..4459
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4519..4535)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4519..4535)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4532..4554)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4543..4559
                     /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)."
     promoter        complement(join(4567..4573,4574..4591,4592..4597))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4612..4633
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4750..4767)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4921..5509)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5001..5020)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5680..6471,6472..6540))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6303..6322
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6541..6645)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggaattc
      901 accaccatgg tgaccggcta ccggctgttc gaggagattc tctccggact taagtctcgg
      961 cgagcgctcg actccatggg ggagaaaccc gggaccaggg tcttcaagaa atcgagtcct
     1021 aactgcaagc tcaccgtgta cctgggcaag cgggactttg tggaccacct ggacaaagtg
     1081 gatcctgtag atggtgtggt gctagtggac ccggactact tgaaggaccg caaagtgttt
     1141 gtgaccctca cctgcgcctt tcgctatggc cgcgaagacc tggacgtgct gggcttgtcc
     1201 ttccgcaaag acctgttcat cgccaactac caggccttcc ccccaacacc caacccaccc
     1261 ccgccccccc cccgcctgca ggagcggctg ctgaggaagc tgggccagca tgcccacccc
     1321 ttttttttca caatacccca gaacctgccc tgctccgtca ccctgcagcc aggcccagag
     1381 gatacgggga aggcctgcgg ggtcgacttt gagattcgag ccttctgcgc caaatcacta
     1441 gaagagaaaa gccacaaacg gaactctgtg cgtctggtga tccgaaaggt gcagtttcca
     1501 cccggccccg aaaccacacg ccacttcctc atgtctgacc ggtccctgca ccttgaggct
     1561 tcactggaca aggagctgta ctaccacggg gagcccctca atgtcaatgt ccacgtcacc
     1621 aataactcca ccaagactgt caagaagatc aaagtctctg tgagacagta cgccgacatc
     1681 tgcctcttca gcaccgccca gtacaagtgc cccgtggctc aggtcgaaca agatgaccag
     1741 gtgtcgccca gttccacgtt ctgtaaggtg tacaccataa ccccgctgct cagcaacaac
     1801 cgggagaagc gcggcctcgc tctagatggg aagctcaagc acgaggacac caacctggcc
     1861 tcgagcacca ttgtgaagga gggtgccaac aaggaggtgc tggggatact ggtgtcctac
     1921 agggtcaagg tgaagctggt ggtgtcacga ggcggggatg tctcagtgga gctgccattt
     1981 gttctcatgc accccaagcc ccatgatcac atcgcccttc ccaggcccca gtcagctgtt
     2041 cctgaaacag atgcccccgt ggacaccaac ctcattgaat ttgaaaccaa ctatgccaca
     2101 gacgacgaca tcgtgttcga agactttgcc cggctgcggc tcaaggggct gaaggacgag
     2161 gactatgacg accagttctg tatcgatgat tacaaggatg acgacgataa gtagaagctt
     2221 ggccctattc tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct
     2281 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     2341 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     2401 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     2461 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     2521 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2581 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2641 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2701 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2761 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2821 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2881 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2941 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     3001 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3061 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3121 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3181 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3241 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3301 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     3361 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     3421 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     3481 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     3541 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     3601 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     3661 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     3721 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     3781 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     3841 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     3901 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     3961 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     4021 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     4081 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     4141 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     4201 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     4261 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4321 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     4381 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4441 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     4501 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4561 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4621 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4681 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4741 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4801 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4861 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4921 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4981 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5041 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5101 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5161 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5221 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5281 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5341 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     5401 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5461 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5521 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5581 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5641 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5701 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5761 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5821 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5881 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5941 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6001 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6061 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6121 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6181 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6241 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6301 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     6361 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6421 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6481 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6541 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6601 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6661 agtgccacct gacgtc
//
