LOCUS       40924_42024        8124 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pT-GEM(2), complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8124)
  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 8124)
  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 8124)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8124)
  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..8124
                     /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..311
                     /label=linker
                     /note="linker"
     CDS             318..371
                     /codon_start=1
                     /label=T2A
                     /note="2A peptide from Thosea asigna virus capsid protein"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             372..3014
                     /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     3521..3554
                     /label=loxP
                     /note="loxP"
     protein_bind    3521..3554
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             3786..4460
                     /codon_start=1
                     /label=DsRed1
                     /note="wild-type DsRed"
                     /translation="MRSSKNVIKEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     intron          4540..4605
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4735..4755
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     polyA_signal    5027..5161
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(5168..5201)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(5208..5307)
                     /label=phage phi-C31 attP
                     /note="attachment site of phage phi-C31"
     misc_feature    5306..5325
                     /label=MCS
                     /note="MCS"
     promoter        complement(5338..5356)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5377..5393)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5401..5417)
                     /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(5425..5455)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5470..5491)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5779..6367)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6541..7398)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7399..7503)
                     /label=AmpR promoter
     rep_origin      7530..7985
                     /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 tccgcaggtc gggaggttcc ggtggaagcg
      301 gaggtagcgg cggatccgag ggccgcggca gcctgctgac ctgcggcgat gtggaggaga
      361 accccgggcc catgaagctg ctgagcagca tcgagcaggc ctgcgatatc tgccgcctga
      421 agaagctgaa gtgcagcaag gagaagccca agtgcgccaa gtgcctgaag aacaactggg
      481 agtgccgcta cagccccaag accaagcgca gccccctgac ccgcgcccac ctgaccgagg
      541 tggagagccg cctggagcgc ctggagcagc tgttcctgct gatcttcccc cgcgaggatc
      601 tggatatgat cctgaagatg gatagcctgc aggatatcaa ggccctgctg accggcctgt
      661 tcgtgcagga taacgtgaac aaggatgccg tgaccgatcg cctggccagc gtggagaccg
      721 atatgcccct gaccctgcgc cagcaccgca tcagcgccac cagcagcagc gaggagagca
      781 gcaacaaggg ccagcgccag ctgaccgtga gcatcgatag cgccgcccac cacgataaca
      841 gcaccatccc cctggatttc atgccccgcg atgccctgca cggcttcgat tggagcgagg
      901 aggatgatat gagcgatggc ctgcccttcc tgaagaccga tcccaacaac aacggcttct
      961 tcggcgatgg cagcctgctg tgcatcctgc gcagcatcgg cttcaagccc gagaactaca
     1021 ccaacagcaa cgtgaaccgc ctgcccacca tgatcaccga tcgctacacc ctggccagcc
     1081 gcagcaccac cagccgcctg ctgcagagct acctgaacaa cttccacccc tactgcccca
     1141 tcgtgcacag ccccaccctg atgatgctgt acaacaacca gatcgagatc gccagcaagg
     1201 atcagtggca gatcctgttc aactgcatcc tggccatcgg cgcctggtgc atcgagggcg
     1261 agagcaccga tatcgatgtg ttctactacc agaacgccaa gagccacctg accagcaagg
     1321 tgttcgagag cggcagcatc atcctggtga ccgccctgca cctgctgagc cgctacaccc
     1381 agtggcgcca gaagaccaac accagctaca acttccacag cttcagcatc cgcatggcca
     1441 tcagcctggg cctgaaccgc gatctgccca gcagcttcag cgatagcagc atcctggagc
     1501 agcgccgccg catctggtgg agcgtgtaca gctgggagat ccagctgagc ctgctgtacg
     1561 gccgcagcat ccagctgagc cagaacacca tcagcttccc cagcagcgtg gatgatgtgc
     1621 agcgcaccac caccggcccc accatctacc acggcatcat cgagaccgcc cgcctgctgc
     1681 aggtgttcac caagatctac gagctggata agaccgtgac cgccgagaag agccccatct
     1741 gcgccaagaa gtgcctgatg atctgcaacg agatcgagga ggtgagccgc caggccccca
     1801 agttcctgca gatggatatc agcaccaccg ccctgaccaa cctgctgaag gagcacccct
     1861 ggctgagctt cacccgcttc gagctgaagt ggaagcagct gagcctgatc atctacgtgc
     1921 tgcgcgattt cttcaccaac ttcacccaga agaagagcca gctggagcag gatcagaacg
     1981 atcaccagag ctacgaggtg aagcgctgca gcatcatgct gagcgatgcc gcccagcgca
     2041 ccgtgatgag cgtgagcagc tacatggata accacaacgt gaccccctac ttcgcctgga
     2101 actgcagcta ctacctgttc aacgccgtgc tggtgcccat caagaccctg ctgagcaaca
     2161 gcaagagcaa cgccgagaac aacgagaccg cccagctgct gcagcagatc aacaccgtgc
     2221 tgatgctgct gaagaagctg gccaccttca agatccagac ctgcgagaag tacatccagg
     2281 tgctggagga ggtgtgcgcc cccttcctgc tgagccagtg cgccatcccc ctgccccaca
     2341 tcagctacaa caacagcaac ggcagcgcca tcaagaacat cgtgggcagc gccaccatcg
     2401 cccagtaccc caccctgccc gaggagaacg tgaacaacat cagcgtgaag tacgtgagcc
     2461 ccggcagcgt gggacccagc cccgtgcccc tgaagagcgg cgccagcttc agcgatctgg
     2521 tgaagctgct gagcaaccgc ccccccagcc gcaacagccc cgtgaccatc ccccgcagca
     2581 cccccagcca ccgcagcgtg acccccttcc tgggccagca gcagcagctg cagagcctgg
     2641 tgcccctgac ccccagcgcc ctgttcggcg gcgccaactt caaccagagc ggcaacatcg
     2701 ccgatagcag cctgagcttc accttcacca acagcagcaa cggccccaac ctgatcacca
     2761 cccagaccaa cagccaggcc ctgagccagc ccatcgccag cagcaacgtg cacgataact
     2821 tcatgaacaa cgagatcacc gccagcaaga tcgatgatgg caacaacagc aagcccctga
     2881 gccccggctg gaccgatcag accgcctaca acgccttcgg catcaccacc ggcatgttca
     2941 acaccaccac catggatgat gtgtacaact acctgttcga tgatgaggat acccccccca
     3001 accccaagaa ggagtaaaat gaatcgtaga tactgaaaaa ccccgcaagt tcacttcaac
     3061 tgtgcatcgt gcaccatctc aatttctttc atttatacat cgttttgcct tcttttatgt
     3121 aactatactc ctctaagttt caatcttggc catgtaacct ctgatctata gaatttttta
     3181 aatgactaga attaatgccc atcttttttt tggacctaaa ttcttcatga aaatatatta
     3241 cgagggctta ttcagaagct tatcgatacc gtcgactaaa gccaaataga aattattcag
     3301 ttctggctta agtttttaaa agtgatatta tttatttggt tgtaaccaac caaaagaatg
     3361 taaataacta atacataatt atgttagttt taagttagca acaaattgat tttagctata
     3421 ttagctactt ggttaataaa tagaatatat ttatttaaag ataattcgtt tttattgtca
     3481 gggagtgagt ttgcttaaaa actcgtttgg atccgacgtc ataacttcgt atagcataca
     3541 ttatacgaag ttatcctagg ggatctaatt caattagaga ctaattcaat tagagctaat
     3601 tcaattagga tccaagctta tcgatttcga accctcgacc gccggagtat aaatagaggc
     3661 gcttcgtcta cggagcgaca attcaattca aacaagcaaa gtgaacacgt cgctaagcga
     3721 aagctaagca aataaacaag cgcagctgaa caagctaaac aatcggacta gagccggtcg
     3781 ccaccatgag gtcttccaag aatgttatca aggagttcat gaggtttaag gttcgcatgg
     3841 aaggaacggt caatgggcac gagtttgaaa tagaaggcga aggagagggg aggccatacg
     3901 aaggccacaa taccgtaaag cttaaggtaa ccaagggggg acctttgcca tttgcttggg
     3961 atattttgtc accacaattt cagtatggaa gcaaggtata tgtcaagcac cctgccgaca
     4021 taccagacta taaaaagctg tcatttcctg aaggatttaa atgggaaagg gtcatgaact
     4081 ttgaagacgg tggcgtcgtt actgtaaccc aggattccag tttgcaggat ggctgtttca
     4141 tctacaaggt caagttcatt ggcgtgaact ttccttccga tggacctgtt atgcaaaaga
     4201 agacaatggg ctgggaagcc agcactgagc gtttgtatcc tcgtgatggc gtgttgaaag
     4261 gagagattca taaggctctg aagctgaaag acggtggtca ttacctagtt gagttcaaaa
     4321 gtatttacat ggcaaagaag cctgtgcagc taccagggta ctactatgtt gactccaaac
     4381 tggatataac aagccacaac gaagactata caatcgttga gcagtatgaa agaaccgagg
     4441 gacgccacca tctgttcctt taggatcttt gtgaaggaac cttacttctg tggtgtgaca
     4501 taattggaca aactacctac agagatttaa agctctaagg taaatataaa atttttaagt
     4561 gtataatgtg ttaaactact gattctaatt gtttgtgtat tttagattcc aacctatgga
     4621 actgatgaat gggagcagtg gtggaatgcc tttaatgagg aaaacctgtt ttgctcagaa
     4681 gaaatgccat ctagtgatga tgaggctact gctgactctc aacattctac tcctccaaaa
     4741 aagaagagaa aggtagaaga ccccaaggac tttccttcag aattgctaag ttttttgagt
     4801 catgctgtgt ttagtaatag aactcttgct tgctttgcta tttacaccac aaaggaaaaa
     4861 gctgcactgc tatacaagaa aattatggaa aaatatttga tgtatagtgc cttgactaga
     4921 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     4981 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     5041 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     5101 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     5161 cgctagcata acttcgtata gcatacatta tacgaagtta tctcgagaac cccttgtgtc
     5221 atgtcggcga ccctacgccc ccaactgaga gaactcaaag gttaccccag ttggggcact
     5281 actcccgaaa accgcttctg acctgggggc gcgccggtac cactagtggg gaggttccct
     5341 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5401 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5461 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5521 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5581 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5641 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5701 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5761 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5821 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5881 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5941 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     6001 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     6061 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6121 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6181 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6241 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6301 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6361 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6421 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6481 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6541 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6601 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6661 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6721 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6781 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6841 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6901 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6961 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     7021 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     7081 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7141 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7201 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7261 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7321 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7381 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7441 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7501 gcgcacattt ccccgaaaag tgccacctga cgcgccctgt agcggcgcat taagcgcggc
     7561 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     7621 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     7681 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     7741 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     7801 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     7861 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     7921 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     7981 aatttgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     8041 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     8101 gccagggttt tcccagtcac gacg
//
