LOCUS       sequence-488537-        6697 bp    DNA     circular SYN 07-OCT-2025
DEFINITION  Expression of mutated bovine arrestin-3 (I385A, V386A, F387A) tagged
            with SmBiT and Flag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6697)
  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 6697)
  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: pSmBtA3-3AF
FEATURES             Location/Qualifiers
     source          1..6697
                     /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"
     rep_origin      2564..2992
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2651..2670)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2861..2882
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3001..3021)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3006..3335
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3186..3321
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3248..3267
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3402..4196
                     /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(3456..3475)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4066..4085
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4370..4491
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4407..4426)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4461..4480
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4540..4556)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4540..4556)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4553..4575)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4564..4580
                     /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(4588..4594,4595..4612,4613..4618))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4633..4654
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4771..4788)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4942..5530)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5022..5041)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5701..6492,6493..6561))
                     /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     6324..6343
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6562..6666)
                     /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 gccgcggctg 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 tcctttctcg ccacgttcgc cggctttccc
     2701 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2761 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2821 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2881 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2941 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     3001 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3061 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3121 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3181 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3241 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3301 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3361 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     3421 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3481 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     3541 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     3601 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     3661 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     3721 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     3781 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     3841 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     3901 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     3961 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4021 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4081 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4141 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4201 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     4261 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4321 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4381 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4441 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4501 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4561 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4621 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4681 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4741 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4801 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4861 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4921 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4981 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5041 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5101 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5161 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5221 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5281 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5341 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5401 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5461 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5521 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5581 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5641 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5701 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5761 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5821 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5881 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5941 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6001 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6061 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6121 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6181 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6241 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6301 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6361 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6421 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6481 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6541 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6601 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6661 tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
