LOCUS       40924_40452        6896 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pSIREN_U6-shRNA_FF3-CMV-iRFP, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6896)
  AUTHORS   Li Y, Jiang Y, Chen H, Liao W, Li Z, Weiss R, Xie Z.
  TITLE     Modular construction of mammalian gene circuits using TALE 
            transcriptional repressors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6896)
  AUTHORS   Li Y, Jiang Y, Chen H, Liao W.
  TITLE     Direct Submission
  JOURNAL   Submitted (03-SEP-2014) Bioinformatics Division/Center for Synthetic
REFERENCE   3  (bases 1 to 6896)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6896)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (03-SEP-2014) Bioinformatics Division/Center for Synthetic "
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..6896
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     78..94
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        102..120
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    186..219
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    229..477
                     /label=Homo sapiens U6 promoter
                     /note="Homo sapiens U6 promoter"
     misc_feature    484..536
                     /label=shRNA-FF3
                     /note="shRNA-FF3"
     terminator      complement(635..729)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     enhancer        801..1104
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1105..1308
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             1361..2308
                     /codon_start=1
                     /label=iRFP713
                     /note="phytochrome-based near-infrared fluorescent protein,
                     originally termed iRFP (Filonov et al., 2011)"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     polyA_signal    2326..2446
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2465..3121)
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     protein_bind    complement(3150..3183)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3198..3643
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             3644..5062
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        5133..5237
                     /label=AmpR promoter
     CDS             5238..6095
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6269..6857
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
       61 attaccgcct tacgcgtgta aaacgacggc cagtagatct gtaatacgac tcactatagg
      121 gcgctagctg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
      181 gccgcataac ttcgtatagc atacattata cgaagttatc agtcgacggg caggaagagg
      241 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
      301 agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
      361 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
      421 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgagga
      481 tccgcgatat gggctgaata caaattcaga ctttgtattc agcccatatc gtttttgaat
      541 tctctagacc attcgtttgg cgcgcgggcc cagtaggtaa gtgaacatgg tcatagctgt
      601 ttcctaggag atcctggtca tgactagtgc ttggattctc accaataaaa aacgcccggc
      661 ggcaaccgag cgttctgaac aaatccagat ggagttctga ggtcattact ggatctatca
      721 acaggagtcc aagcgagctc tagttattaa tagtaatcaa ttacggggtc attagttcat
      781 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      841 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      901 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      961 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     1021 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     1081 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     1141 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     1201 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     1261 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac
     1321 cgtcagatcc gctagcgcta ccggactcag atccaccggt atggctgaag gatccgtcgc
     1381 caggcagcct gacctcttga cctgcgacga tgagccgatc catatccccg gtgccatcca
     1441 accgcatgga ctgctgctcg ccctcgccgc cgacatgacg atcgttgccg gcagcgacaa
     1501 ccttcccgaa ctcaccggac tggcgatcgg cgccctgatc ggccgctctg cggccgatgt
     1561 cttcgactcg gagacgcaca accgtctgac gatcgccttg gccgagcccg gggcggccgt
     1621 cggagcaccg atcactgtcg gcttcacgat gcgaaaggac gcaggcttca tcggctcctg
     1681 gcatcgccat gatcagctca tcttcctcga gctcgagcct ccccagcggg acgtcgccga
     1741 gccgcaggcg ttcttccgcc gcaccaacag cgccatccgc cgcctgcagg ccgccgaaac
     1801 cttggaaagc gcctgcgccg ccgcggcgca agaggtgcgg aagattaccg gcttcgatcg
     1861 ggtgatgatc tatcgcttcg cctccgactt cagcggcgaa gtgatcgcag aggatcggtg
     1921 cgccgaggtc gagtcaaaac taggcctgca ctatcctgcc tcaaccgtgc cggcgcaggc
     1981 ccgtcggctc tataccatca acccggtacg gatcattccc gatatcaatt atcggccggt
     2041 gccggtcacc ccagacctca atccggtcac cgggcggccg attgatctta gcttcgccat
     2101 cctgcgcagc gtctcgcccg tccatctgga attcatgcgc aacataggca tgcacggcac
     2161 gatgtcgatc tcgattttgc gcggcgagcg actgtgggga ttgatcgttt gccatcaccg
     2221 aacgccgtac tacgtcgatc tcgatggccg ccaagcctgc gagctagtcg cccaggttct
     2281 ggcctggcag atcggcgtga tggaagagtg acaattgttg ttgttaactt gtttattgca
     2341 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     2401 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttgagc tcgatatcaa
     2461 attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga
     2521 catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt
     2581 cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg
     2641 ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat
     2701 tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg
     2761 aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg
     2821 tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct
     2881 caccgtcttt cattgccata cgaaattccg gatgagcatt catcaggcgg gcaagaatgt
     2941 gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa
     3001 tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat
     3061 gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca
     3121 ttttagcttc cttagctcct gaaagatcca taacttcgta tagcatacat tatacgaagt
     3181 tatgcggccg cgacgtccac atatacctgc cgttcactat tatttagtga aatgagatat
     3241 tatgatattt tctgaattgt gattaaaaag gcaactttat gcccatgcaa cagaaactat
     3301 aaaaaataca gagaatgaaa agaaacagat agatttttta gttctttagg cccgtagtct
     3361 gcaaatcctt ttatgatttt ctatcaaaca aaagaggaaa atagaccagt tgcaatccaa
     3421 acgagagtct aatagaatga ggtcgaaaag taaatcgcgc gggtttgtta ctgataaagc
     3481 aggcaagacc taaaatgtgt aaagggcaaa gtgtatactt tggcgtcacc ccttacatat
     3541 tttaggtctt tttttattgt gcgtaactaa cttgccatct tcaaacagga gggctggaag
     3601 aagcagaccg ctaacacagt acataaaaaa ggagacatga acgatgaaca tcaaaaagtt
     3661 tgcaaaacaa gcaacagtat taacctttac taccgcactg ctggcaggag gcgcaactca
     3721 agcgtttgcg aaagaaacga accaaaagcc atataaggaa acatacggca tttcccatat
     3781 tacacgccat gatatgctgc aaatccctga acagcaaaaa aatgaaaaat atcaagttcc
     3841 tgagttcgat tcgtccacaa ttaaaaatat ctcttctgca aaaggcctgg acgtttggga
     3901 cagctggcca ttacaaaacg ctgacggcac tgtcgcaaac tatcacggct accacatcgt
     3961 ctttgcatta gccggagatc ctaaaaatgc ggatgacaca tcgatttaca tgttctatca
     4021 aaaagtcggc gaaacttcta ttgacagctg gaaaaacgct ggccgcgtct ttaaagacag
     4081 cgacaaattc gatgcaaatg attctatcct aaaagaccaa acacaagaat ggtcaggttc
     4141 agccacattt acatctgacg gaaaaatccg tttattctac actgatttct ccggtaaaca
     4201 ttacggcaaa caaacactga caactgcaca agttaacgta tcagcatcag acagctcttt
     4261 gaacatcaac ggtgtagagg attataaatc aatctttgac ggtgacggaa aaacgtatca
     4321 aaatgtacag cagttcatcg atgaaggcaa ctacagctca ggcgacaacc atacgctgag
     4381 agatcctcac tacgtagaag ataaaggcca caaatactta gtatttgaag caaacactgg
     4441 aactgaagat ggctaccaag gcgaagaatc tttatttaac aaagcatact atggcaaaag
     4501 cacatcattc ttccgtcaag aaagtcaaaa acttctgcaa agcgataaaa aacgcacggc
     4561 tgagttagca aacggcgctc tcggtatgat tgagctaaac gatgattaca cactgaaaaa
     4621 agtgatgaaa ccgctgattg catctaacac agtaacagat gaaattgaac gcgcgaacgt
     4681 ctttaaaatg aacggcaaat ggtacctgtt cactgactcc cgcggatcaa aaatgacgat
     4741 tgacggcatt acgtctaacg atatttacat gcttggttat gtttctaatt ctttaactgg
     4801 cccatacaag ccgctgaaca aaactggcct tgtgttaaaa atggatcttg atcctaacga
     4861 tgtaaccttt acttactcac acttcgctgt acctcaagcg aaaggaaaca atgtcgtgat
     4921 tacaagctat atgacaaaca gaggattcta cgcagacaaa caatcaacgt ttgcgcctag
     4981 cttcctgctg aacatcaaag gcaagaaaac atctgttgtc aaagacagca tccttgaaca
     5041 aggacaatta acagttaaca aataaaaacg caaaagaaaa tgccgatatc ctattggcat
     5101 tgacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5161 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5221 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5281 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5341 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5401 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5461 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5521 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5581 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5641 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5701 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5761 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5821 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5881 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5941 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6001 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6061 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6121 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6181 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6241 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6301 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6361 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6421 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6481 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6541 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6601 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6661 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6721 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6781 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6841 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttt
//
