LOCUS       sequence-492662-        6292 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mutated bovine arrestin-3 (D291R) tagged with SmBiT 
            and Flag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6292)
  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 6292)
  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: "Cells."; 
            date: "2023-06-6"; volume: "12(12):1563. doi"; pages: " 
            10.3390/cells12121563"
COMMENT     Sequence Label: pSmBtA3-DRF
FEATURES             Location/Qualifiers
     source          1..6292
                     /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             2209..2232
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2250..2268)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2251..2268)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2288..2305)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2294..2518
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2651..2670)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2712..2922
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        2734..2930
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2781..2916
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2843..2862
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2997..3791
                     /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(3051..3070)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3661..3680
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3965..4086
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4002..4021)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4056..4075
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4135..4151)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4135..4151)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4148..4170)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4159..4175
                     /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(4183..4189,4190..4207,4208..4213))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4228..4249
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4366..4383)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4537..5125)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4617..4636)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5296..6087,6088..6156))
                     /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     5919..5938
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6157..6261)
                     /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 cctaggcaag cgggactttg tggaccacct ggacaaagtg
     1081 gaccctgtag atggtgtggt gctagtggat ccggactact tgaaggaccg caaagtgttt
     1141 gtgaccctca cgtgcgcctt tcgctatggc cgcgaagacc tggacgtcct gggcttgtcc
     1201 ttccgcaaag acctgttcat cgccaactac caggccttcc ctccaacacc taacccacct
     1261 agaccaccta cgcgtctgca ggagcggctg ctgaggaagc tgggccagca tgcccacccc
     1321 ttttttttca caatacccca gaacctgccc tgctccgtca ccctacagcc aggcccagag
     1381 gatacgggga aggcttgcgg ggtcgacttt gagattcgag ccttctgcgc caaatcacta
     1441 gaagagaaaa gccacaaacg gaactccgta cgtctggtga tccgaaaggt gcagtttgcc
     1501 ccagagaaac cagggcccca gccttcagct gaaaccacac gccacttcct catgtctgac
     1561 cggtccctgc accttgaggc ttcactggac aaggagctgt actaccacgg ggagcccctc
     1621 aatgtcaatg tccacgtcac caataactcc accaagactg tcaagaagat caaagtctct
     1681 gtgagacagt acgccgacat ctgcctcttc agcaccgccc agtacaagtg ccccgtggct
     1741 caggtcgaac aagatgacca ggtgtcgccc agttccacgt tctgtaaggt gtacaccata
     1801 accccgctgc tcagcaacaa ccgggagaag cgcggcctcg ctctacgtgg gaagctcaag
     1861 cacgaggaca ccaacctggc ctcgagcacc attgtgaagg agggtgccaa caaggaggtg
     1921 ctggggatac tggtgtccta cagggtcaag gtgaagctgg tggtgtcacg aggcggggat
     1981 gtctcagtgg agctgccatt tgttctcatg caccccaagc cccatgatca catcgccctt
     2041 cccaggcccc agtcagctgt tcctgaaaca gatgcccccg tggacaccaa cctcattgaa
     2101 tttgaaacca actatgccac agacgacgac atcgtgttcg aagactttgc ccggctgcgg
     2161 ctcaaggggc tgaaggacga ggactatgac gaccagttct gtatcgatga ttacaaggat
     2221 gacgacgata agtagaagct tggccctatt ctatagtgtc acctaaatgc tagagctcgc
     2281 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2341 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2401 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2461 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2521 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2581 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2641 ctagcgcccg ctcctttcgc tttcttccct tccagtcagc aaccaggtgt ggaaagtccc
     2701 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     2761 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     2821 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     2881 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     2941 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     3001 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3061 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     3121 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     3181 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     3241 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     3301 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     3361 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     3421 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     3481 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     3541 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     3601 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     3661 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     3721 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     3781 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     3841 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     3901 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     3961 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4021 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4081 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4141 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4201 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4261 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4321 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4381 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4441 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4501 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4561 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4621 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4681 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4741 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4801 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4861 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4921 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4981 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5041 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5101 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5161 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5221 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5281 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5341 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5401 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5461 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5521 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5581 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5641 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5701 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5761 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5821 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5881 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5941 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6001 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6061 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6121 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6181 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6241 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
