LOCUS       40924_42019        8125 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT-GEM(1), complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8125)
  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 8125)
  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 8125)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8125)
  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..8125
                     /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..312
                     /label=linker
                     /note="linker"
     CDS             319..372
                     /codon_start=1
                     /label=T2A
                     /note="2A peptide from Thosea asigna virus capsid protein"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             373..3015
                     /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     3522..3555
                     /label=loxP
                     /note="loxP"
     protein_bind    3522..3555
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             3787..4461
                     /codon_start=1
                     /label=DsRed1
                     /note="wild-type DsRed"
                     /translation="MRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     intron          4541..4606
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4736..4756
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     polyA_signal    5028..5162
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5169..5202)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(5209..5308)
                     /label=phage phi-C31 attP
                     /note="attachment site of phage phi-C31"
     misc_feature    5309..5328
                     /label=MCS
                     /note="MCS"
     promoter        complement(5339..5357)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5378..5394)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5402..5418)
                     /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(5426..5456)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5471..5492)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5780..6368)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6542..7399)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7400..7504)
                     /label=AmpR promoter
     rep_origin      7531..7986
                     /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 gcggaggttc cggtggaagc
      301 ggaggtagcg gcggatccga gggccgcggc agcctgctga cctgcggcga tgtggaggag
      361 aaccccgggc ccatgaagct gctgagcagc atcgagcagg cctgcgatat ctgccgcctg
      421 aagaagctga agtgcagcaa ggagaagccc aagtgcgcca agtgcctgaa gaacaactgg
      481 gagtgccgct acagccccaa gaccaagcgc agccccctga cccgcgccca cctgaccgag
      541 gtggagagcc gcctggagcg cctggagcag ctgttcctgc tgatcttccc ccgcgaggat
      601 ctggatatga tcctgaagat ggatagcctg caggatatca aggccctgct gaccggcctg
      661 ttcgtgcagg ataacgtgaa caaggatgcc gtgaccgatc gcctggccag cgtggagacc
      721 gatatgcccc tgaccctgcg ccagcaccgc atcagcgcca ccagcagcag cgaggagagc
      781 agcaacaagg gccagcgcca gctgaccgtg agcatcgata gcgccgccca ccacgataac
      841 agcaccatcc ccctggattt catgccccgc gatgccctgc acggcttcga ttggagcgag
      901 gaggatgata tgagcgatgg cctgcccttc ctgaagaccg atcccaacaa caacggcttc
      961 ttcggcgatg gcagcctgct gtgcatcctg cgcagcatcg gcttcaagcc cgagaactac
     1021 accaacagca acgtgaaccg cctgcccacc atgatcaccg atcgctacac cctggccagc
     1081 cgcagcacca ccagccgcct gctgcagagc tacctgaaca acttccaccc ctactgcccc
     1141 atcgtgcaca gccccaccct gatgatgctg tacaacaacc agatcgagat cgccagcaag
     1201 gatcagtggc agatcctgtt caactgcatc ctggccatcg gcgcctggtg catcgagggc
     1261 gagagcaccg atatcgatgt gttctactac cagaacgcca agagccacct gaccagcaag
     1321 gtgttcgaga gcggcagcat catcctggtg accgccctgc acctgctgag ccgctacacc
     1381 cagtggcgcc agaagaccaa caccagctac aacttccaca gcttcagcat ccgcatggcc
     1441 atcagcctgg gcctgaaccg cgatctgccc agcagcttca gcgatagcag catcctggag
     1501 cagcgccgcc gcatctggtg gagcgtgtac agctgggaga tccagctgag cctgctgtac
     1561 ggccgcagca tccagctgag ccagaacacc atcagcttcc ccagcagcgt ggatgatgtg
     1621 cagcgcacca ccaccggccc caccatctac cacggcatca tcgagaccgc ccgcctgctg
     1681 caggtgttca ccaagatcta cgagctggat aagaccgtga ccgccgagaa gagccccatc
     1741 tgcgccaaga agtgcctgat gatctgcaac gagatcgagg aggtgagccg ccaggccccc
     1801 aagttcctgc agatggatat cagcaccacc gccctgacca acctgctgaa ggagcacccc
     1861 tggctgagct tcacccgctt cgagctgaag tggaagcagc tgagcctgat catctacgtg
     1921 ctgcgcgatt tcttcaccaa cttcacccag aagaagagcc agctggagca ggatcagaac
     1981 gatcaccaga gctacgaggt gaagcgctgc agcatcatgc tgagcgatgc cgcccagcgc
     2041 accgtgatga gcgtgagcag ctacatggat aaccacaacg tgacccccta cttcgcctgg
     2101 aactgcagct actacctgtt caacgccgtg ctggtgccca tcaagaccct gctgagcaac
     2161 agcaagagca acgccgagaa caacgagacc gcccagctgc tgcagcagat caacaccgtg
     2221 ctgatgctgc tgaagaagct ggccaccttc aagatccaga cctgcgagaa gtacatccag
     2281 gtgctggagg aggtgtgcgc ccccttcctg ctgagccagt gcgccatccc cctgccccac
     2341 atcagctaca acaacagcaa cggcagcgcc atcaagaaca tcgtgggcag cgccaccatc
     2401 gcccagtacc ccaccctgcc cgaggagaac gtgaacaaca tcagcgtgaa gtacgtgagc
     2461 cccggcagcg tgggacccag ccccgtgccc ctgaagagcg gcgccagctt cagcgatctg
     2521 gtgaagctgc tgagcaaccg cccccccagc cgcaacagcc ccgtgaccat cccccgcagc
     2581 acccccagcc accgcagcgt gacccccttc ctgggccagc agcagcagct gcagagcctg
     2641 gtgcccctga cccccagcgc cctgttcggc ggcgccaact tcaaccagag cggcaacatc
     2701 gccgatagca gcctgagctt caccttcacc aacagcagca acggccccaa cctgatcacc
     2761 acccagacca acagccaggc cctgagccag cccatcgcca gcagcaacgt gcacgataac
     2821 ttcatgaaca acgagatcac cgccagcaag atcgatgatg gcaacaacag caagcccctg
     2881 agccccggct ggaccgatca gaccgcctac aacgccttcg gcatcaccac cggcatgttc
     2941 aacaccacca ccatggatga tgtgtacaac tacctgttcg atgatgagga tacccccccc
     3001 aaccccaaga aggagtaaaa tgaatcgtag atactgaaaa accccgcaag ttcacttcaa
     3061 ctgtgcatcg tgcaccatct caatttcttt catttataca tcgttttgcc ttcttttatg
     3121 taactatact cctctaagtt tcaatcttgg ccatgtaacc tctgatctat agaatttttt
     3181 aaatgactag aattaatgcc catctttttt ttggacctaa attcttcatg aaaatatatt
     3241 acgagggctt attcagaagc ttatcgatac cgtcgactaa agccaaatag aaattattca
     3301 gttctggctt aagtttttaa aagtgatatt atttatttgg ttgtaaccaa ccaaaagaat
     3361 gtaaataact aatacataat tatgttagtt ttaagttagc aacaaattga ttttagctat
     3421 attagctact tggttaataa atagaatata tttatttaaa gataattcgt ttttattgtc
     3481 agggagtgag tttgcttaaa aactcgtttg gatccgacgt cataacttcg tatagcatac
     3541 attatacgaa gttatcctag gggatctaat tcaattagag actaattcaa ttagagctaa
     3601 ttcaattagg atccaagctt atcgatttcg aaccctcgac cgccggagta taaatagagg
     3661 cgcttcgtct acggagcgac aattcaattc aaacaagcaa agtgaacacg tcgctaagcg
     3721 aaagctaagc aaataaacaa gcgcagctga acaagctaaa caatcggact agagccggtc
     3781 gccaccatga ggtcttccaa gaatgttatc aaggagttca tgaggtttaa ggttcgcatg
     3841 gaaggaacgg tcaatgggca cgagtttgaa atagaaggcg aaggagaggg gaggccatac
     3901 gaaggccaca ataccgtaaa gcttaaggta accaaggggg gacctttgcc atttgcttgg
     3961 gatattttgt caccacaatt tcagtatgga agcaaggtat atgtcaagca ccctgccgac
     4021 ataccagact ataaaaagct gtcatttcct gaaggattta aatgggaaag ggtcatgaac
     4081 tttgaagacg gtggcgtcgt tactgtaacc caggattcca gtttgcagga tggctgtttc
     4141 atctacaagg tcaagttcat tggcgtgaac tttccttccg atggacctgt tatgcaaaag
     4201 aagacaatgg gctgggaagc cagcactgag cgtttgtatc ctcgtgatgg cgtgttgaaa
     4261 ggagagattc ataaggctct gaagctgaaa gacggtggtc attacctagt tgagttcaaa
     4321 agtatttaca tggcaaagaa gcctgtgcag ctaccagggt actactatgt tgactccaaa
     4381 ctggatataa caagccacaa cgaagactat acaatcgttg agcagtatga aagaaccgag
     4441 ggacgccacc atctgttcct ttaggatctt tgtgaaggaa ccttacttct gtggtgtgac
     4501 ataattggac aaactaccta cagagattta aagctctaag gtaaatataa aatttttaag
     4561 tgtataatgt gttaaactac tgattctaat tgtttgtgta ttttagattc caacctatgg
     4621 aactgatgaa tgggagcagt ggtggaatgc ctttaatgag gaaaacctgt tttgctcaga
     4681 agaaatgcca tctagtgatg atgaggctac tgctgactct caacattcta ctcctccaaa
     4741 aaagaagaga aaggtagaag accccaagga ctttccttca gaattgctaa gttttttgag
     4801 tcatgctgtg tttagtaata gaactcttgc ttgctttgct atttacacca caaaggaaaa
     4861 agctgcactg ctatacaaga aaattatgga aaaatatttg atgtatagtg ccttgactag
     4921 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     4981 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     5041 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5101 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     5161 tcgctagcat aacttcgtat agcatacatt atacgaagtt atctcgagaa ccccttgtgt
     5221 catgtcggcg accctacgcc cccaactgag agaactcaaa ggttacccca gttggggcac
     5281 tactcccgaa aaccgcttct gacctggggg cgcgccggta ccactagtgg ggaggttccc
     5341 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5401 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5461 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5521 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5581 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5641 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5701 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5761 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5821 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5881 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5941 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6001 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6061 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6121 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6181 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6241 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6301 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6361 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6421 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6481 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6541 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6601 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6661 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6721 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6781 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6841 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6901 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6961 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7021 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7081 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7141 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7201 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7261 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7321 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7381 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7441 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7501 cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg
     7561 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     7621 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     7681 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     7741 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     7801 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     7861 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     7921 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     7981 caatttgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
     8041 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
     8101 cgccagggtt ttcccagtca cgacg
//
