LOCUS       40924_42014        8123 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT-GEM(0), complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8123)
  AUTHORS   Diao F, Ironfield H, Luan H, Diao F, Shropshire WC, Ewer J, Marr E, 
            Potter CJ, Landgraf M, White BH.
  TITLE     Plug-and-Play Genetic Access to Drosophila Cell Types using 
            Exchangeable Exon Cassettes
  JOURNAL   Cell Rep 10 (8), 1410-1421 (2015)
  PUBMED    25732830
REFERENCE   2  (bases 1 to 8123)
  AUTHORS   Diao F, Luan H, White BH.
  TITLE     Direct Submission
  JOURNAL   Submitted (22-JAN-2015) Laboratory of Molecular Biology, NIMH, NIH, 
            35 Convent Dr, Bethesda, MD 20850, USA
REFERENCE   3  (bases 1 to 8123)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8123)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep"; 
            date: "2015"; volume: "10"; issue: "8"; pages: "1410-1421"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (22-JAN-2015) Laboratory of Molecular Biology, NIMH, NIH, 35 Convent
            Dr, Bethesda, MD 20850, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Assembly Method       :: Vector NTI v. Version 11
            Sequencing Technology :: Illumina
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..8123
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     3..19
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        26..44
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    52..71
                     /label=MCS
                     /note="MCS"
     protein_bind    72..171
                     /label=phage phi-C31 attP
                     /note="attachment site of phage phi-C31"
     misc_feature    178..277
                     /label=MHC splice acceptor site
                     /note="MHC splice acceptor site"
     misc_feature    278..310
                     /label=linker
                     /note="linker"
     CDS             317..370
                     /codon_start=1
                     /label=T2A
                     /note="2A peptide from Thosea asigna virus capsid protein"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             371..3013
                     /codon_start=1
                     /label=GAL4
                     /note="GAL4 transcriptional activator"
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSIDSAAHHDNSTIPLDF
                     MPRDALHGFDWSEEDDMSDGLPFLKTDPNNNGFFGDGSLLCILRSIGFKPENYTNSNVN
                     RLPTMITDRYTLASRSTTSRLLQSYLNNFHPYCPIVHSPTLMMLYNNQIEIASKDQWQI
                     LFNCILAIGAWCIEGESTDIDVFYYQNAKSHLTSKVFESGSIILVTALHLLSRYTQWRQ
                     KTNTSYNFHSFSIRMAISLGLNRDLPSSFSDSSILEQRRRIWWSVYSWEIQLSLLYGRS
                     IQLSQNTISFPSSVDDVQRTTTGPTIYHGIIETARLLQVFTKIYELDKTVTAEKSPICA
                     KKCLMICNEIEEVSRQAPKFLQMDISTTALTNLLKEHPWLSFTRFELKWKQLSLIIYVL
                     RDFFTNFTQKKSQLEQDQNDHQSYEVKRCSIMLSDAAQRTVMSVSSYMDNHNVTPYFAW
                     NCSYYLFNAVLVPIKTLLSNSKSNAENNETAQLLQQINTVLMLLKKLATFKIQTCEKYI
                     QVLEEVCAPFLLSQCAIPLPHISYNNSNGSAIKNIVGSATIAQYPTLPEENVNNISVKY
                     VSPGSVGPSPVPLKSGASFSDLVKLLSNRPPSRNSPVTIPRSTPSHRSVTPFLGQQQQL
                     QSLVPLTPSALFGGANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     misc_recomb     3520..3553
                     /label=loxP
                     /note="loxP"
     protein_bind    3520..3553
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             3785..4459
                     /codon_start=1
                     /label=DsRed1
                     /note="wild-type DsRed"
                     /translation="MRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     intron          4539..4604
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4734..4754
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     polyA_signal    5026..5160
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5167..5200)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(5207..5306)
                     /label=phage phi-C31 attP
                     /note="attachment site of phage phi-C31"
     misc_feature    5307..5326
                     /label=MCS
                     /note="MCS"
     promoter        complement(5337..5355)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5376..5392)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5400..5416)
                     /label=lac operator
                     /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(5424..5454)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5469..5490)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5778..6366)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6540..7397)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7398..7502)
                     /label=AmpR promoter
     rep_origin      7529..7984
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgacc caccggtgca
       61 tgcgcggccg ccccaggtca gaagcggttt tcgggagtag tgccccaact ggggtaacct
      121 ttgagttctc tcagttgggg gcgtagggtc gccgacatga cacaaggggt ttctagaagt
      181 cgatccaaca tggcgacttg tcccatcccc ggcatgttta aatatactaa ttattcttga
      241 actaatttta atcaaccgat ttatctctct tccgcaggtg ggaggttccg gtggaagcgg
      301 aggtagcggc ggatccgagg gccgcggcag cctgctgacc tgcggcgatg tggaggagaa
      361 ccccgggccc atgaagctgc tgagcagcat cgagcaggcc tgcgatatct gccgcctgaa
      421 gaagctgaag tgcagcaagg agaagcccaa gtgcgccaag tgcctgaaga acaactggga
      481 gtgccgctac agccccaaga ccaagcgcag ccccctgacc cgcgcccacc tgaccgaggt
      541 ggagagccgc ctggagcgcc tggagcagct gttcctgctg atcttccccc gcgaggatct
      601 ggatatgatc ctgaagatgg atagcctgca ggatatcaag gccctgctga ccggcctgtt
      661 cgtgcaggat aacgtgaaca aggatgccgt gaccgatcgc ctggccagcg tggagaccga
      721 tatgcccctg accctgcgcc agcaccgcat cagcgccacc agcagcagcg aggagagcag
      781 caacaagggc cagcgccagc tgaccgtgag catcgatagc gccgcccacc acgataacag
      841 caccatcccc ctggatttca tgccccgcga tgccctgcac ggcttcgatt ggagcgagga
      901 ggatgatatg agcgatggcc tgcccttcct gaagaccgat cccaacaaca acggcttctt
      961 cggcgatggc agcctgctgt gcatcctgcg cagcatcggc ttcaagcccg agaactacac
     1021 caacagcaac gtgaaccgcc tgcccaccat gatcaccgat cgctacaccc tggccagccg
     1081 cagcaccacc agccgcctgc tgcagagcta cctgaacaac ttccacccct actgccccat
     1141 cgtgcacagc cccaccctga tgatgctgta caacaaccag atcgagatcg ccagcaagga
     1201 tcagtggcag atcctgttca actgcatcct ggccatcggc gcctggtgca tcgagggcga
     1261 gagcaccgat atcgatgtgt tctactacca gaacgccaag agccacctga ccagcaaggt
     1321 gttcgagagc ggcagcatca tcctggtgac cgccctgcac ctgctgagcc gctacaccca
     1381 gtggcgccag aagaccaaca ccagctacaa cttccacagc ttcagcatcc gcatggccat
     1441 cagcctgggc ctgaaccgcg atctgcccag cagcttcagc gatagcagca tcctggagca
     1501 gcgccgccgc atctggtgga gcgtgtacag ctgggagatc cagctgagcc tgctgtacgg
     1561 ccgcagcatc cagctgagcc agaacaccat cagcttcccc agcagcgtgg atgatgtgca
     1621 gcgcaccacc accggcccca ccatctacca cggcatcatc gagaccgccc gcctgctgca
     1681 ggtgttcacc aagatctacg agctggataa gaccgtgacc gccgagaaga gccccatctg
     1741 cgccaagaag tgcctgatga tctgcaacga gatcgaggag gtgagccgcc aggcccccaa
     1801 gttcctgcag atggatatca gcaccaccgc cctgaccaac ctgctgaagg agcacccctg
     1861 gctgagcttc acccgcttcg agctgaagtg gaagcagctg agcctgatca tctacgtgct
     1921 gcgcgatttc ttcaccaact tcacccagaa gaagagccag ctggagcagg atcagaacga
     1981 tcaccagagc tacgaggtga agcgctgcag catcatgctg agcgatgccg cccagcgcac
     2041 cgtgatgagc gtgagcagct acatggataa ccacaacgtg accccctact tcgcctggaa
     2101 ctgcagctac tacctgttca acgccgtgct ggtgcccatc aagaccctgc tgagcaacag
     2161 caagagcaac gccgagaaca acgagaccgc ccagctgctg cagcagatca acaccgtgct
     2221 gatgctgctg aagaagctgg ccaccttcaa gatccagacc tgcgagaagt acatccaggt
     2281 gctggaggag gtgtgcgccc ccttcctgct gagccagtgc gccatccccc tgccccacat
     2341 cagctacaac aacagcaacg gcagcgccat caagaacatc gtgggcagcg ccaccatcgc
     2401 ccagtacccc accctgcccg aggagaacgt gaacaacatc agcgtgaagt acgtgagccc
     2461 cggcagcgtg ggacccagcc ccgtgcccct gaagagcggc gccagcttca gcgatctggt
     2521 gaagctgctg agcaaccgcc cccccagccg caacagcccc gtgaccatcc cccgcagcac
     2581 ccccagccac cgcagcgtga cccccttcct gggccagcag cagcagctgc agagcctggt
     2641 gcccctgacc cccagcgccc tgttcggcgg cgccaacttc aaccagagcg gcaacatcgc
     2701 cgatagcagc ctgagcttca ccttcaccaa cagcagcaac ggccccaacc tgatcaccac
     2761 ccagaccaac agccaggccc tgagccagcc catcgccagc agcaacgtgc acgataactt
     2821 catgaacaac gagatcaccg ccagcaagat cgatgatggc aacaacagca agcccctgag
     2881 ccccggctgg accgatcaga ccgcctacaa cgccttcggc atcaccaccg gcatgttcaa
     2941 caccaccacc atggatgatg tgtacaacta cctgttcgat gatgaggata ccccccccaa
     3001 ccccaagaag gagtaaaatg aatcgtagat actgaaaaac cccgcaagtt cacttcaact
     3061 gtgcatcgtg caccatctca atttctttca tttatacatc gttttgcctt cttttatgta
     3121 actatactcc tctaagtttc aatcttggcc atgtaacctc tgatctatag aattttttaa
     3181 atgactagaa ttaatgccca tctttttttt ggacctaaat tcttcatgaa aatatattac
     3241 gagggcttat tcagaagctt atcgataccg tcgactaaag ccaaatagaa attattcagt
     3301 tctggcttaa gtttttaaaa gtgatattat ttatttggtt gtaaccaacc aaaagaatgt
     3361 aaataactaa tacataatta tgttagtttt aagttagcaa caaattgatt ttagctatat
     3421 tagctacttg gttaataaat agaatatatt tatttaaaga taattcgttt ttattgtcag
     3481 ggagtgagtt tgcttaaaaa ctcgtttgga tccgacgtca taacttcgta tagcatacat
     3541 tatacgaagt tatcctaggg gatctaattc aattagagac taattcaatt agagctaatt
     3601 caattaggat ccaagcttat cgatttcgaa ccctcgaccg ccggagtata aatagaggcg
     3661 cttcgtctac ggagcgacaa ttcaattcaa acaagcaaag tgaacacgtc gctaagcgaa
     3721 agctaagcaa ataaacaagc gcagctgaac aagctaaaca atcggactag agccggtcgc
     3781 caccatgagg tcttccaaga atgttatcaa ggagttcatg aggtttaagg ttcgcatgga
     3841 aggaacggtc aatgggcacg agtttgaaat agaaggcgaa ggagagggga ggccatacga
     3901 aggccacaat accgtaaagc ttaaggtaac caagggggga cctttgccat ttgcttggga
     3961 tattttgtca ccacaatttc agtatggaag caaggtatat gtcaagcacc ctgccgacat
     4021 accagactat aaaaagctgt catttcctga aggatttaaa tgggaaaggg tcatgaactt
     4081 tgaagacggt ggcgtcgtta ctgtaaccca ggattccagt ttgcaggatg gctgtttcat
     4141 ctacaaggtc aagttcattg gcgtgaactt tccttccgat ggacctgtta tgcaaaagaa
     4201 gacaatgggc tgggaagcca gcactgagcg tttgtatcct cgtgatggcg tgttgaaagg
     4261 agagattcat aaggctctga agctgaaaga cggtggtcat tacctagttg agttcaaaag
     4321 tatttacatg gcaaagaagc ctgtgcagct accagggtac tactatgttg actccaaact
     4381 ggatataaca agccacaacg aagactatac aatcgttgag cagtatgaaa gaaccgaggg
     4441 acgccaccat ctgttccttt aggatctttg tgaaggaacc ttacttctgt ggtgtgacat
     4501 aattggacaa actacctaca gagatttaaa gctctaaggt aaatataaaa tttttaagtg
     4561 tataatgtgt taaactactg attctaattg tttgtgtatt ttagattcca acctatggaa
     4621 ctgatgaatg ggagcagtgg tggaatgcct ttaatgagga aaacctgttt tgctcagaag
     4681 aaatgccatc tagtgatgat gaggctactg ctgactctca acattctact cctccaaaaa
     4741 agaagagaaa ggtagaagac cccaaggact ttccttcaga attgctaagt tttttgagtc
     4801 atgctgtgtt tagtaataga actcttgctt gctttgctat ttacaccaca aaggaaaaag
     4861 ctgcactgct atacaagaaa attatggaaa aatatttgat gtatagtgcc ttgactagag
     4921 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     4981 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     5041 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     5101 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     5161 gctagcataa cttcgtatag catacattat acgaagttat ctcgagaacc ccttgtgtca
     5221 tgtcggcgac cctacgcccc caactgagag aactcaaagg ttaccccagt tggggcacta
     5281 ctcccgaaaa ccgcttctga cctgggggcg cgccggtacc actagtgggg aggttccctt
     5341 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     5401 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     5461 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5521 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5581 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5641 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5701 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5761 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5821 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5881 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5941 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6001 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6061 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6121 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6181 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     6241 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6301 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6361 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6421 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6481 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6541 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6601 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6661 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6721 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6781 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6841 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6901 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6961 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     7021 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7081 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7141 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7201 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7261 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     7321 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     7381 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     7441 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7501 cgcacatttc cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg
     7561 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     7621 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     7681 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     7741 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     7801 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     7861 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     7921 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     7981 atttgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     8041 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     8101 ccagggtttt cccagtcacg acg
//
