LOCUS       Exported                6316 bp ds-DNA     circular SYN 10-MAR-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6316)
  AUTHORS   MIT
  TITLE     Direct Submission
  JOURNAL   Exported Sunday, Feb 13, 2022 from SnapGene 5.2.5
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6316
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     misc_feature    588..746
                     /label=LacZ
     primer_bind     812..831
                     /label=BII(R)
     misc_feature    874..1110
                     /label=5prime_from_parent
     repeat_region   874..936
                     /label=5prime_repeat_unit
                     /label=nonstandard type:  repeat_unit
     misc_feature    874..886
                     /label=Perfect_repeat_5
     misc_feature    887..917
                     /label=31bp spacer
     misc_feature    918..936
                     /label=subterminal_repeat_5
     enhancer        1112..1343
                     /label=core insul
     primer_bind     1335..1369
                     /label=TRE-fp-56
     misc_feature    1348..1350
                     /label=AAV D' element
     promoter        1361..1675
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1369..1387
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1405..1423
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1440..1458
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1476..1494
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1512..1530
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1547..1565
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1583..1601
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1640..1658
                     /label=TRE_281_fsp (for pBOB-TRE-nOB)
                     /note="1.3.3"
     CDS             1677..1679
                     /codon_start=1
                     /label=ORF frame 3
                     /label=ORF frame 3(1)
                     /translation="M"
     regulatory      1689..1698
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1695..2414
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1821..1838)
                     /label=EGFP_fsp_52
                     /note="9.5.9"
     primer_bind     2183..2200
                     /label=EGFP_rsp_51
                     /note="9.6.1"
     primer_bind     2233..2249
                     /label=Venus_555_fsp
                     /note="4.3.6"
     primer_bind     2383..2399
                     /label=EGFP_689_fsp
                     /note="9.1.1"
     primer_bind     complement(2393..2440)
                     /label=EGFP-rp-55
     primer_bind     complement(2393..2410)
                     /label=EGFP_1_fsp_for sTpE
                     /note="1.7.8"
     primer_bind     2395..2411
                     /label=EGFP_701_fsp
                     /note="1.5.1"
     CDS             2415..2417
                     /codon_start=1
                     /label=EGFP
                     /translation=""
     misc_feature    2415..2417
                     /label=TagRFP-T
     primer_bind     2418..2448
                     /label=pB_BamHI_WPRE_fp_64.1
     misc_feature    2418..2422
                     /label=NotI
     misc_feature    2418..2421
                     /label=MCS
     misc_feature    2422
                     /label=NotI GC/GGCCGC
     misc_feature    2423..3014
                     /label=WPRE (match to WHV8 genome, J04514)
     misc_feature    2423..2440
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     misc_feature    3015
                     /label=EagI C/GGCCG
     misc_feature    3016..3239
                     /label=BGH pA
     primer_bind     complement(3224..3255)
                     /label=pB_BGHpA_63.5
     misc_feature    3240
                     /label=SpeI A/CTAGT
     misc_feature    3241..3247
                     /label=MCS
     enhancer        complement(3265..3495)
                     /label=core insul(1)
     misc_feature    join(3495..6316,1..1111)
                     /label=pXL_BAC_II_homology
     misc_feature    3496..3804
                     /label=3prime_from_parent
     repeat_region   3770..3804
                     /label=3'TR
                     /label=nonstandard type:  repeat_unit
     misc_feature    3770..3788
                     /label=subterminal_repeat_3
     misc_feature    3789..3791
                     /label=3bp_spacer
     misc_feature    3792..3804
                     /label=perfect_repeat_3
     misc_feature    join(4279..6316,1..746)
                     /label=pUC backbone
     primer_bind     complement(4323..4342)
                     /label=pLac(F)
     CDS             complement(5459..6316)
                     /label=Amp
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc aacgcgtctc gagtttactc cctatcagtg
     1381 atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgatgt cgagtttact
     1441 ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat
     1501 gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttatccc tatcagtgat
     1561 agagaacgta tgtcgagttt actccctatc agtgatagag aacgtatgtc gaggtaggcg
     1621 tgtacggtgg gaggcctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag
     1681 aattcgccgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     1741 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     1801 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     1861 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     1921 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     1981 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     2041 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     2101 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     2161 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     2221 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     2281 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     2341 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     2401 agctgtacaa gtaataagat ccaatcaacc tctggattac aaaatttgtg aaagattgac
     2461 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     2521 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     2581 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     2641 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     2701 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     2761 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct
     2821 gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     2881 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     2941 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     3001 cgcctccccg cctgctgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     3061 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     3121 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3181 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatgga
     3241 gatccgcggc ccattggtat ggctttttcc ccgtatcccc ccaggtgtct gcaggctcaa
     3301 agagcagcga gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct
     3361 gtccccgcac gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg
     3421 ggcggctcgc tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg
     3481 gggggggctg tccctgatat ctataacaag aaaatatata tataataagt tatcacgtaa
     3541 gtagaacatg aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa
     3601 gataatcatg cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac
     3661 cgcattgaca agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac
     3721 agcgacggat tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat
     3781 tttacgcaga ctatctttct agggttaatc tagctgcatc aggatcatat cgtcgggtct
     3841 tttttccggc tcagtcatcg cccaagctgg cgctatctgg gcatcgggga ggaagaagcc
     3901 cgtgcctttt cccgcgaggt tgaagcggca tggaaagagt ttgccgagga tgactgctgc
     3961 tgcattgacg ttgagcgaaa acgcacgttt accatgatga ttcgggaagg tgtggccatg
     4021 cacgccttta acggtgaact gttcgttcag gccacctggg ataccagttc gtcgcggctt
     4081 ttccggacac agttccggat ggtcagcccg aagcgcatca gcaacccgaa caataccggc
     4141 gacagccgga actgccgtgc cggtgtgcag attaatgaca gcggtgcggc gctgggatat
     4201 tacgtcagcg aggacgggta tcctggctgg atgccgcaga aatggacatg gataccccgt
     4261 gagttacccg gcgggcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4321 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4381 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4441 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4501 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4561 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4621 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4681 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4741 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4801 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4861 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4921 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4981 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5041 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5101 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     5161 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5221 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5281 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5341 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5401 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5461 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5521 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5581 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5641 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5701 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5761 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5821 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5881 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5941 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6001 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6061 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6121 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6181 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6241 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6301 aatgttgaat actcat
//
