LOCUS       sequence-387508-        5349 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mRNA synthesis of rapalog-induced cell adhesion molecule.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5349)
  AUTHORS   Usami C, Inomata H
  TITLE     Rapalog-induced cell adhesion molecule inhibits mesoderm migration 
            in Xenopus embryos by increasing frequency of adhesion to the 
            ectoderm.
  JOURNAL   Genes Cells. 2022 Jun;27(6):436-450. doi: 10.1111/gtc.12937. Epub 
            2022 May 4.
  PUBMED    35437867
REFERENCE   2  (bases 1 to 5349)
  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: "10.1111/gtc.12937";
            journalName: "Genes Cells"; date: "2022-06"; volume: "27"; issue: 
            "6"; pages: "436-450"
COMMENT     Sequence Label: pCS2-SP-FRB (T2098L) -sfGFP-TM-V5
FEATURES             Location/Qualifiers
     source          1..5349
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        38..56
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     38..55
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             178..456
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             join(457..459,460..462,463..1173)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(502..523)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1110..1131
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1330..1371
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    1404..1538
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1428..1447)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1482..1501
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1651..1671)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1651..1669)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     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
     rep_origin      complement(3842..4297)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3979..4000)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4191..4210
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(4368..5349,1..3)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 aaccgccatt ctgcctgggg acgtcggagc aagcttgatt taggtgacac tatagaatac
       61 aagctacttg ttctttttgc aggatccgcc atgaactcct tctccacaag cgccttcggt
      121 ccagttgcct tctccctggg cctgctcctg gtgttgcctg ctgccttccc tgccccaatc
      181 ctctggcatg agatgtggca tgaaggcctg gaagaggcat ctcgtttgta ctttggggaa
      241 aggaacgtga aaggcatgtt tgaggtgctg gagcccttgc atgctatgat ggaacggggc
      301 ccccagactc tgaaggaaac atcctttaat caggcctatg gtcgagattt aatggaggcc
      361 caagagtggt gcaggaagta catgaaatca gggaatgtca aggacctcct ccaagcctgg
      421 gacctctatt atcatgtgtt ccgacgaatc tcaaagatgg tgagcaaggg cgaggagctg
      481 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
      541 agcgtgcgtg gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc
      601 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
      661 gtgcagtgct tcagccgcta ccccgaccac atgaagcggc acgacttctt caagtccgcc
      721 atgcccgaag gctacgtcca ggagcgcacc atctctttca aggacgacgg cacctacaag
      781 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
      841 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctttaacagc
      901 cacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc
      961 cgccacaacg ttgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     1021 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgtcctg
     1081 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1141 gggatcactc atggcatgga cgagctgtac aaggagctgt tttgggcact ggtcgtggtt
     1201 gctggagtcc tgttttgtta tggcttgcta gtgacagtgg ctctttgtgt tatctggaca
     1261 aatagtagaa ggaacagagg tggctctggc ggtggcggat cgggtggagg ttcgtccgga
     1321 ggccccgggg gtaagcctat ccctaaccct ctcctcggtc tcgattctac gtagtctaga
     1381 actatagtga gtcgtattac gtagatccag acatgataag atacattgat gagtttggac
     1441 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     1501 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     1561 ttatgtttca ggttcagggg gaggtgtggg aggtttttta attcgcggcc gcggcgccaa
     1621 tgcattgggc ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     1681 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     1741 catacgagcc gggagcataa 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 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     3901 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     3961 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     4021 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     4081 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     4141 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     4201 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     4261 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
     4321 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagtc gaccgccaat
     4381 tcaatatggc gtatatggac tcatgccaat tcaatatggt ggatctggac ctgtgccaat
     4441 tcaatatggc gtatatggac tcgtgccaat tcaatatggt ggatctggac cccagccaat
     4501 tcaatatggc ggacttggca ccatgccaat tcaatatggc ggacctggca ctgtgccaac
     4561 tggggagggg tctacttggc acggtgccaa gtttgaggag gggtcttggc cctgtgccaa
     4621 gtccgccata ttgaattggc atggtgccaa taatggcggc catattggct atatgccagg
     4681 atcaatatat aggcaatatc caatatggcc ctatgccaat atggctattg gccaggttca
     4741 atactatgta ttggccctat gccatatagt attccatata tgggttttcc tattgacgta
     4801 gatagcccct cccaatgggc ggtcccatat accatatatg gggcttccta ataccgccca
     4861 tagccactcc cccattgacg tcaatggtct ctatatatgg tctttcctat tgacgtcata
     4921 tgggcggtcc tattgacgta tatggcgcct cccccattga cgtcaattac ggtaaatggc
     4981 ccgcctggct caatgcccat tgacgtcaat aggaccaccc accattgacg tcaatgggat
     5041 ggctcattgc ccattcatat ccgttctcac gccccctatt gacgtcaatg acggtaaatg
     5101 gcccacttgg cagtacatca atatctatta atagtaactt ggcaagtaca ttactattgg
     5161 aagtacgcca gggtacattg gcagtactcc cattgacgtc aatggcggta aatggcccgc
     5221 gatggctgcc aagtacatcc ccattgacgt caatggggag gggcaatgac gcaaatgggc
     5281 gttccattga cgtaaatggg cggtaggcgt gcctaatggg aggtctatat aagcaatgct
     5341 cgtttaggg
//
