LOCUS       sequence-227217-        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
FEATURES             Location/Qualifiers
     source          1..5990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        113..492
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        493..696
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     647..667
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     741..760
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        741..759
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      791..800
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             800..847
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     promoter        complement(1421..1439)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1422..1439)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1459..1476)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1465..1689
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1735..2163
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1822..1841)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2032..2053
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2172..2192)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2177..2506
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2357..2492
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2419..2438
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2573..3367
                     /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(2627..2646)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3237..3256
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3541..3662
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3578..3597)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3632..3651
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3711..3727)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3711..3727)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3724..3746)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3735..3751
                     /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(3759..3765,3766..3783,3784..3789))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3804..3825
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3942..3959)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4113..4701)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4193..4212)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4872..5663,5664..5732))
                     /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     5495..5514
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5733..5837)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5912..5931)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
       61 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga
      121 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      181 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      241 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      301 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      361 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      421 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      481 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      541 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      601 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      661 aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact
      721 gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc ttggtaccga
      781 gctcggatcc gccaccatgg actacaagga cgacgatgac aaggactaca aggacgacga
      841 tgacaagaat tctcgtgagt ataagctagt cgttcttggc tctggaggcg ttggaaaatc
      901 tgctctgact gtacagtttg ttcaaggaat ttttgttgaa aaatatgatc ctacgataga
      961 agattcttat agaaagcaag ttgaagtaga tgcacagcag tgtatgcttg aaatcttgga
     1021 tactgcagga acggaacaat tcacagcaat gagggatttg tacatgaaaa atggacaagg
     1081 ctttgcgtta gtttattcca tcacagcaca gtccacattt aatgatttac aagatctgag
     1141 agaacagata cttcgagtta aagacacgga tgatgttcca atgattctgg ttggtaataa
     1201 gtgtgacttg gaagatgaaa gagttgtagg aaaggaacaa ggtcaaaatc tagcaagaca
     1261 gtggaacaac tgtgcattct tagaatcctc tgcaaaatca aaaataaatg ttaatgagat
     1321 cttttatgac ctagtgcgac aaattaacag aaaaactcca gtgcctggga aggcccgcaa
     1381 aaagtcatca tgtcagctgc tttaatctag agggccctat tctatagtgt cacctaaatg
     1441 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     1501 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     1561 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     1621 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     1681 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     1741 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1801 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1861 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1921 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1981 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2041 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2101 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2161 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     2221 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     2281 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     2341 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     2401 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     2461 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     2521 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     2581 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     2641 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     2701 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     2761 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     2821 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     2881 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     2941 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     3001 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     3061 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     3121 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     3181 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     3241 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     3301 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     3361 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     3421 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     3481 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     3541 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3601 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3661 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     3721 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     3781 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     3841 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3901 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3961 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4021 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4081 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4141 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4201 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4261 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4321 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4381 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4441 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4501 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4561 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4621 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4681 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4741 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4801 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4861 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4921 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4981 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     5041 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5101 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5161 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5221 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5281 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5341 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5401 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5461 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5521 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5581 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5641 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5701 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5761 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5821 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     5881 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5941 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
//
