LOCUS       sequence-227287-        5990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of bovine Rap1b.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5990)
  AUTHORS   Takahashi M, Dillon TJ, Liu C, Kariya Y, Wang Z, Stork PJ
  TITLE     Protein kinase A-dependent phosphorylation of Rap1 regulates its 
            membrane localization and cell migration.
  JOURNAL   J Biol Chem. 2013 Sep 27;288(39):27712-23. doi: 
            10.1074/jbc.M113.466904. Epub 2013 Aug 14.
  PUBMED    23946483
REFERENCE   2  (bases 1 to 5990)
  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"; doi: 
            "10.1074/jbc.M113.466904"; journalName: "J Biol Chem"; date: 
            "2013-09-27- 27"; volume: "288"; issue: "39"; pages: "27712-23"
COMMENT     Sequence Label: Flag-Rap1b S179AS180A
FEATURES             Location/Qualifiers
     source          1..5990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        123..502
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        503..706
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     657..677
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     751..770
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        751..769
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      801..810
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             810..857
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     promoter        complement(1431..1449)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1432..1449)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1469..1486)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1475..1699
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1745..2173
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1832..1851)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2042..2063
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2182..2202)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2187..2516
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2367..2502
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2429..2448
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2583..3377
                     /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(2637..2656)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3247..3266
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3551..3672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3588..3607)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3642..3661
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3721..3737)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3721..3737)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3734..3756)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3745..3761
                     /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(3769..3775,3776..3793,3794..3799))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3814..3835
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3952..3969)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4123..4711)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4203..4222)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4882..5673,5674..5742))
                     /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     5505..5524
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5743..5847)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5922..5941)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
       61 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      121 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      181 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      241 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      301 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      361 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      421 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      481 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      541 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      601 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      661 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
      721 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
      781 ttggtaccga gctcggatcc gccaccatgg actacaagga cgacgatgac aaggactaca
      841 aggacgacga tgacaagaat tctcgtgagt ataagctagt cgttcttggc tctggaggcg
      901 ttggaaaatc tgctctgact gtacagtttg ttcaaggaat ttttgttgaa aaatatgatc
      961 ctacgataga agattcttat agaaagcaag ttgaagtaga tgcacagcag tgtatgcttg
     1021 aaatcttgga tactgcagga acggaacaat tcacagcaat gagggatttg tacatgaaaa
     1081 atggacaagg ctttgcgtta gtttattcca tcacagcaca gtccacattt aatgatttac
     1141 aagatctgag agaacagata cttcgagtta aagacacgga tgatgttcca atgattctgg
     1201 ttggtaataa gtgtgacttg gaagatgaaa gagttgtagg aaaggaacaa ggtcaaaatc
     1261 tagcaagaca gtggaacaac tgtgcattct tagaatcctc tgcaaaatca aaaataaatg
     1321 ttaatgagat cttttatgac ctagtgcgac agattaacag aaaaactcca gtgcctggga
     1381 aggcccgcaa aaaggcggca tgtcagctgc tttaatctag agggccctat tctatagtgt
     1441 cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     1501 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1561 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     1621 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     1681 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     1741 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     1801 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     1861 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     1921 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     1981 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2041 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2101 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2161 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2221 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2281 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2341 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     2401 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     2461 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     2521 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     2581 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     2641 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     2701 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     2761 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     2821 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     2881 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     2941 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3001 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3061 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     3121 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3181 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3241 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     3301 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     3361 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     3421 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     3481 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     3541 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3601 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3661 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     3721 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3781 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     3841 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     3901 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     3961 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4021 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4081 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4141 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4201 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4261 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4321 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4381 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4441 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4501 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4561 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4621 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4681 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4741 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4801 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4861 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4921 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4981 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5041 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5101 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5161 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5221 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5281 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5341 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5401 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5461 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5521 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5581 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5641 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5701 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5761 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5821 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     5881 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     5941 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
//
