LOCUS       sequence-488747-        6679 bp    DNA     circular SYN 08-OCT-2025
DEFINITION  Expression of mutated bovine arrestin-3 (L394Stop) tagged with SmBiT
            and Flag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6679)
  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 6679)
  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: pSmBtA3-TrF
FEATURES             Location/Qualifiers
     source          1..6679
                     /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"
     promoter        complement(2232..2250)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2233..2250)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2270..2287)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2276..2500
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2546..2974
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2633..2652)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2843..2864
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2983..3003)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2988..3317
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3168..3303
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3230..3249
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3384..4178
                     /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(3438..3457)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4048..4067
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4352..4473
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4389..4408)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4443..4462
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4522..4538)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4522..4538)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4535..4557)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4546..4562
                     /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(4570..4576,4577..4594,4595..4600))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4615..4636
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4753..4770)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4924..5512)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5004..5023)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5683..6474,6475..6543))
                     /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     6306..6325
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6544..6648)
                     /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 ctctagatgg 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 ccggtagcgg
     2161 ctcaaggggc tgaaggacga ggactatgac gaccagttct gtatcgatta gggctggaag
     2221 cttggcccta ttctatagtg tcacctaaat gctagagctc gctgatcagc ctcgactgtg
     2281 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2341 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2401 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2461 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2521 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2581 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2641 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2701 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2761 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2821 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2881 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2941 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     3001 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3061 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3121 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3181 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3241 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3301 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     3361 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     3421 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3481 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3541 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     3601 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3661 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3721 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3781 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3841 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3901 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3961 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     4021 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     4081 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     4141 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     4201 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     4261 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4321 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     4381 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4441 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4501 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4561 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4621 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4681 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4741 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4801 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4861 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4921 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4981 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5041 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5101 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5161 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5221 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5281 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5341 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5401 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5461 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5521 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5581 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5641 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5701 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5761 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5821 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5881 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5941 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6001 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6061 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6121 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6181 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6241 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6301 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6361 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6421 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6481 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6541 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6601 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6661 aaaagtgcca cctgacgtc
//
