LOCUS       sequence-169605-        7585 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Role of Cdc-42 selective GEF domain from ITSN in immune cell 
            migration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7585)
  AUTHORS   O'Neill PR, Kalyanaraman V, Gautam N
  TITLE     Subcellular optogenetic activation of Cdc42 controls local and 
            distal signaling to drive immune cell migration.
  JOURNAL   Mol Biol Cell. 2016 May 1;27(9):1442-50. doi: 
            10.1091/mbc.E15-12-0832. Epub 2016 Mar 3.
  PUBMED    26941336
REFERENCE   2  (bases 1 to 7585)
  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: 
            "10.1091/mbc.E15-12-0832"; journalName: "Mol Biol Cell"; date: 
            "2016-05-1- 1"; volume: "27"; issue: "9"; pages: "1442-50"
COMMENT     Sequence Label: ITSN-mcherry-sspB
FEATURES             Location/Qualifiers
     source          1..7585
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..32
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(151..171)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        156..485
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      336..471
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     398..417
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             552..1346
                     /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(606..625)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1216..1235
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1520..1641
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1557..1576)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1611..1630
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1690..1706)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1690..1706)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1703..1725)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1714..1730
                     /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(1738..1744,1745..1762,1763..1768))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1783..1804
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1921..1938)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2092..2680)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2172..2191)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2851..3642,3643..3711))
                     /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     3474..3493
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3712..3816)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3891..3910)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4082..4461
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4462..4665
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4616..4636
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4710..4729
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4710..4728
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5873..6580
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6018..6036)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6286..6305
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             join(6656..6862,6863..6865,6866..6985)
                     /codon_start=1
                     /product="SspB from Haemophilus influenzae, with the 
                     mutations Y11K and A15E to disrupt the natural dimerization
                     of the protein and with the mutation R74Q to reduce the 
                     affinity for iLID"
                     /label=SspB micro
                     /note="dimerizes with iLID at moderate affinity to create 
                     iLID micro (Guntas et al., 2015)"
                     /translation="SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQ
                     IVLNLSASATGNLQLTNDFIQFNAQFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     primer_bind     complement(7023..7040)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7029..7253
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(7299..7585,1..142)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7386..7405)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
       61 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
      121 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
      181 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
      241 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
      301 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
      361 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
      421 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
      481 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
      541 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
      601 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
      661 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
      721 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
      781 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
      841 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
      901 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
      961 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     1021 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     1081 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     1141 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     1201 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     1261 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     1321 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     1381 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     1441 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     1501 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     1561 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     1621 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     1681 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     1741 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     1801 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1861 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     1921 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     1981 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2041 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2101 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2161 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2221 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2281 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2341 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2401 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2461 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     2521 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2581 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     2641 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     2701 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     2761 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     2821 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     2881 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     2941 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     3001 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3061 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     3121 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     3181 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     3241 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     3301 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3361 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     3421 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     3481 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     3541 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     3601 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     3661 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     3721 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3781 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     3841 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     3901 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     3961 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     4021 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     4081 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
     4141 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     4201 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     4261 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     4321 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     4381 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     4441 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     4501 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     4561 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     4621 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga
     4681 gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct
     4741 ggctagcgtt taaacttaag cttggtaccg cgggcccgcc accatgttga ccccaactga
     4801 aagaaagcga caaggataca tccacgagct cattgtcacc gaggagaact atgtgaatga
     4861 cctgcagctg gtcacagaga tttttcaaaa acccctgatg gagtctgagc tgctgacaga
     4921 aaaagaggtt gctatgattt ttgtgaactg gaaggagctg attatgtgta atatcaaact
     4981 actaaaagcg ctgagagtcc gcaagaagat gtccggggag aagatgcctg tgaagatgat
     5041 tggagacatc ctgagcgcac agctgccgca catgcagccc tacatccgct tctgcagccg
     5101 ccagctcaac ggggctgccc tgatccagca gaagacggac gaggccccag acttcaagga
     5161 gttcgtcaaa agattggcaa tggatcctcg gtgtaaaggg atgccactct ctagttttat
     5221 actgaagcct atgcaacggg taacaagata cccactgatc attaaaaata tcctggaaaa
     5281 cacccctgaa aaccacccgg accacagcca cttgaagcac gccctggaga aggcggaaga
     5341 gctctgttcc caggtgaacg aaggggtgcg ggagaaggag aactctgacc ggctggagtg
     5401 gatccaggcc cacgtgcagt gtgaaggcct gtctgagcaa cttgtgttca attcagtgac
     5461 caattgcttg gggccgcgca aatttctgca cagtgggaag ctctacaagg ccaagagcaa
     5521 caaggagctg tatggcttcc ttttcaacga cttcctcctg ctgactcaga tcacgaagcc
     5581 tttggggtct tctggcaccg acaaagtctt cagccccaaa tcaaacctgc agtataaaat
     5641 gtataaaaca cctattttcc taaatgaggt tctagtaaaa ttacccaccg acccttctgg
     5701 agacgagccc atcttccaca tctcccacat tgaccgcgtc tatactctcc gagcagaaag
     5761 cataaatgaa aggactgcct gggtgcagaa aatcaaagct gcttctgaac tctacataga
     5821 gactgagaaa aagaaggaat tcagtgctgg atccggtagt ggaagtggtt ccatggtgag
     5881 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     5941 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     6001 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     6061 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     6121 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     6181 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     6241 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     6301 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     6361 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     6421 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     6481 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     6541 gggccgccac tccaccggcg gcatggacga gctgtacaag tccggactca gatctcgagc
     6601 tcaagcttcg aacgaattcg gcattgatct gagcggcctg accctgcagg aattcagctc
     6661 cccgaaacgc cctaagctgc tgcgtgaata ttacgattgg ctggttgata acagctttac
     6721 cccatatctg gtggtggatg ccacatacct gggcgtgaac gtgcccgtgg agtatgtgaa
     6781 agacggtcag atcgtgctga atctgtctgc aagtgcgacc ggcaacctgc aactgacaaa
     6841 tgattttatc cagttcaacg cccagtttaa gggcgtgtct cgtgaactgt atatcccgat
     6901 gggtgccgct ctggccattt acgctcgcga gaacggcgat ggtgtgatgt tcgaaccaga
     6961 agaaatctat gacgagctga atattggtta atctagaggg cccgtttaaa cccgctgatc
     7021 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     7081 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     7141 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     7201 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     7261 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     7321 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     7381 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     7441 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     7501 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     7561 tcgccctttg acgttggagt ccacg
//
