LOCUS       PB-5'-ENGRAM            5681 bp ds-DNA     circular     14-NOV-2021
DEFINITION  .
FEATURES             Location/Qualifiers
     misc_feature    25..49
                     /label="5' gibson overhang"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     misc_feature    53..77
                     /label="3' gibson overhang"
                     /ApEinfo_revcolor=#b1ff67
                     /ApEinfo_fwdcolor=#b1ff67
     misc_feature    226..814
                     /label="ORI (mismatch with STAR by 1)"
                     /ApEinfo_revcolor=#75c6a9
                     /ApEinfo_fwdcolor=#75c6a9
     primer          complement(306..325)
                     /label="062_choi_pe_BR322Ori_rev"
                     /note="sequence: gggaaacgcctggtatcttt"
                     /ApEinfo_revcolor=#c7b0e3
                     /ApEinfo_fwdcolor=#c7b0e3
     CDS             complement(988..1845)
                     /label="Amp"
                     /ApEinfo_revcolor=#84b0dc
                     /ApEinfo_fwdcolor=#84b0dc
     primer_bind     2657..2676
                     /label="BII(R)"
                     /ApEinfo_revcolor=#84b0dc
                     /ApEinfo_fwdcolor=#84b0dc
     primer          2657..2676
                     /label="063_choi_pe_BIIR_PB_fwd"
                     /note="sequence: TTTACAGCGTGATGGAGCAG"
                     /ApEinfo_revcolor=#b4abac
                     /ApEinfo_fwdcolor=#b4abac
     misc_feature    2715..2718
                     /label="TTAA"
                     /ApEinfo_revcolor=#d59687
                     /ApEinfo_fwdcolor=#d59687
     misc_feature    2719..2731
                     /label="13 bp terminal repeat (TR)"
                     /ApEinfo_revcolor=#cd0532
                     /ApEinfo_fwdcolor=#cd0532
     misc_feature    2719..2781
                     /label="5'PB Terminal Repeat"
                     /ApEinfo_revcolor=#0080ff
                     /ApEinfo_fwdcolor=#0080ff
     misc_feature    2732..2762
                     /label="31 bp spacer"
                     /ApEinfo_revcolor=#0080c0
                     /ApEinfo_fwdcolor=#0080c0
     misc_feature    2763..2781
                     /label="19 bp Internal Repeat"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     misc_feature    2957..3188
                     /label="core Insulator"
                     /ApEinfo_revcolor=#d59687
                     /ApEinfo_fwdcolor=#d59687
     primer          3173..3202
                     /label="024_wc_pe_PB-5gibson"
                     /note="sequence: GGGGATACGGGGAAAAGGCCTCCACGGCCA"
                     /ApEinfo_revcolor=#d59687
                     /ApEinfo_fwdcolor=#d59687
     misc_feature    3173..3202
                     /label="5' gibson"
                     /ApEinfo_revcolor=#b7e6d7
                     /ApEinfo_fwdcolor=#b7e6d7
     misc_feature    3202..3202
                     /label="CMV7\promoter"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     misc_feature    3211..3218
                     /label="SbfI"
                     /ApEinfo_revcolor=#f58a5e
                     /ApEinfo_fwdcolor=#f58a5e
     primer          complement(3213..3237)
                     /label="014_wc_tf_p7_amp_rev_nouse"
                     /note="sequence: CATTATATACCCTCTAGTGCCTGCA"
                     /ApEinfo_revcolor=#c7b0e3
                     /ApEinfo_fwdcolor=#c7b0e3
     misc_feature    3219..3272
                     /label="minimal promoter"
                     /ApEinfo_revcolor=#ff00ff
                     /ApEinfo_fwdcolor=#ff00ff
     primer          3248..3307
                     /label="066_wc_tf_csy4-bsmbI-step2-fwd"
                     /note="sequence: TTCCAGCTTGGCAATCCGGTACTGTACCGGTcacttgagacgattaaATGGACAGCAtgc"
                     /ApEinfo_revcolor=#b7e6d7
                     /ApEinfo_fwdcolor=#b7e6d7
     primer          3283..3342
                     /label="065_wc_tf_csy4-bsmbI-step1-fwd"
                     /note="sequence: tgagacgattaaATGGACAGCAtgcgtctcaAGTCCgccaccatggaccactacctcgac"
                     /ApEinfo_revcolor=#b7e6d7
                     /ApEinfo_fwdcolor=#b7e6d7
     regulatory      3319..3328
                     /label="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)"
                     /ApEinfo_revcolor=#d59687
                     /ApEinfo_fwdcolor=#d59687
     CDS             3325..3885
                     /label="Csy4"
                     /ApEinfo_revcolor=#9eafd2
                     /ApEinfo_fwdcolor=#9eafd2
     CDS             3325..3915
                     /label="Translation 3325-3915"
     primer          complement(3886..3915)
                     /label="002_wc_NLS_rev"
                     /note="sequence: tcagactttcctcttcttcttgggggatcc"
                     /ApEinfo_revcolor=#84b0dc
                     /ApEinfo_fwdcolor=#84b0dc
     CDS             3892..3912
                     /label="SV40 NLS"
                     /ApEinfo_revcolor=#b7e6d7
                     /ApEinfo_fwdcolor=#b7e6d7
     misc_feature    3916..4049
                     /label="Triplex modified Lu"
                     /ApEinfo_revcolor=#f58a5e
                     /ApEinfo_fwdcolor=#f58a5e
     primer          complement(4032..4090)
                     /label="067_wc_tf_triplex-3'csy4-gibson-rev"
                     /note="sequence: CGAAGTTATTAGGTCCCTCGACgctgcctatacggcagtgttcgcagaccgatacgaac"
                     /ApEinfo_revcolor=#75c6a9
                     /ApEinfo_fwdcolor=#75c6a9
     misc_feature    4050..4050
                     /label="di-nucleotide change for synthesis"
                     /ApEinfo_revcolor=#d6b295
                     /ApEinfo_fwdcolor=#d6b295
     protein_bind    4050..4068
                     /label="Csy4 site"
                     /ApEinfo_revcolor=#d6b295
                     /ApEinfo_fwdcolor=#d6b295
     primer          complement(4068..4090)
                     /label="068_wc_tf_plsmP_rev"
                     /note="sequence: CGAAGTTATTAGGTCCCTCGACG"
                     /ApEinfo_revcolor=#d59687
                     /ApEinfo_fwdcolor=#d59687
     misc_feature    complement(4069..4090)
                     /label="pLSmP-ass-R-i# sits here"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     primer          complement(4069..4128)
                     /label="072_wc_tf_csy4_pb+3'gibson"
                     /note="sequence: TTTGTAACCATTATAAGCTGCAATAAACAAGTTTAGTTCGAAGTTATTAGGTCCCTCGAC"
                     /ApEinfo_revcolor=#84b0dc
                     /ApEinfo_fwdcolor=#84b0dc
     misc_feature    complement(4091..4130)
                     /label="3'BlastGibson"
                     /ApEinfo_revcolor=#faac61
                     /ApEinfo_fwdcolor=#faac61
     primer          complement(4091..4130)
                     /label="026_wc_pe_PB_3gibson_v2"
                     /note="sequence: TATTTGTAACCATTATAAGCTGCAATAAACAAGTTTAGTT"
                     /ApEinfo_revcolor=#84b0dc
                     /ApEinfo_fwdcolor=#84b0dc
     misc_feature    4096..4226
                     /label="SV40 polyA"
                     /ApEinfo_revcolor=#84b0dc
                     /ApEinfo_fwdcolor=#84b0dc
     primer          complement(4103..4132)
                     /label="004_wc_SV40_rev"
                     /note="sequence: TTTATTTGTAACCATTATAAGCTGCAATAA"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     misc_feature    4475..4705
                     /label="core Insulator"
                     /ApEinfo_revcolor=#ff0080
                     /ApEinfo_fwdcolor=#ff0080
     misc_feature    4980..4998
                     /label="19 bp Internal Repeat(IR)"
                     /ApEinfo_revcolor=#ff0080
                     /ApEinfo_fwdcolor=#ff0080
     misc_feature    4980..5014
                     /label="3' PB Terminal Repeat"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     misc_feature    4999..5001
                     /label="3 bp spacer"
                     /ApEinfo_revcolor=#ffef86
                     /ApEinfo_fwdcolor=#ffef86
     misc_feature    5002..5014
                     /label="13 bp Terminal Repeat (TR)"
                     /ApEinfo_revcolor=#85dae9
                     /ApEinfo_fwdcolor=#85dae9
     misc_feature    5015..5018
                     /label="TTAA"
                     /ApEinfo_revcolor=#d59687
                     /ApEinfo_fwdcolor=#d59687
     misc_feature    complement(5221..5240)
                     /label="Potential primer mismatch"
                     /ApEinfo_revcolor=#c7b0e3
                     /ApEinfo_fwdcolor=#c7b0e3
     primer_bind     complement(5533..5552)
                     /label="pLac(F)"
                     /ApEinfo_revcolor=#84b0dc
                     /ApEinfo_fwdcolor=#84b0dc
     primer          join(5674..5681,1..14)
                     /label="103_am_join_rev1"
                     /note="sequence: CATTAATGAATCGGCCAACGCG"
                     /ApEinfo_revcolor=#ff9ccd
                     /ApEinfo_fwdcolor=#ff9ccd
ORIGIN
        1 AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCTCTTCCG CTTCCTCGCT
       61 CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAGGC
      121 GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGT GAGCAAAAGG
      181 CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG
      241 CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG
      301 ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC
      361 CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA
      421 TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT
      481 GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC
      541 CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG
      601 AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTACAC
      661 TAGAAGGACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAGAGT
      721 TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA
      781 GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG
      841 GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATCAAA
      901 AAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA TCTAAAGTAT
      961 ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC
     1021 GATCTGTCTA TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT
     1081 ACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGACC CACGCTCACC
     1141 GGCTCCAGAT TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC
     1201 TGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG
     1261 TTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT ACAGGCATCG TGGTGTCACG
     1321 CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG
     1381 ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG
     1441 TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT
     1501 CATGCCATCC GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA
     1561 ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGATA ATACCGCGCC
     1621 ACATAGCAGA ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC
     1681 AAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC
     1741 TTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC
     1801 CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT TCCTTTTTCA
     1861 ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT
     1921 TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTAAATT
     1981 GTAAGCGTTA ATATTTTGTT AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT
     2041 AACCAATAGG CCGAAATCGG CAAAATCCCT TATAAATCAA AAGAATAGAC CGAGATAGGG
     2101 TTGAGTGTTG TTCCAGTTTG GAACAAGAGT CCACTATTAA AGAACGTGGA CTCCAACGTC
     2161 AAAGGGCGAA AAACCGTCTA TCAGGGCGAT GGCCCACTAC GTGAACCATC ACCCTAATCA
     2221 AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA
     2281 TTTAGAGCTT GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA
     2341 GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC TGCGCGTAAC CACCACACCC
     2401 GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TCCCATTCGC CATTCAGGCT GCGCAACTGT
     2461 TGGGAAGGGC GATCGGTGCG GGCCTCTTCG CTATTACGCC AGCTGGCGAA AGGGGGATGT
     2521 GCTGCAAGGC GATTAAGTTG GGTAACGCCA GGGTTTTCCC AGTCACGACG TTGTAAAACG
     2581 ACGGCCAGTG AGCGCGCCTC GTTCATTCAC GTTTTTGAAC CCGTGGAGGA CGGGCAGACT
     2641 CGCGGTGCAA ATGTGTTTTA CAGCGTGATG GAGCAGATGA AGATGCTCGA CACGCTGCAG
     2701 AACACGCAGC TAGATTAACC CTAGAAAGAT AATCATATTG TGACGTACGT TAAAGATAAT
     2761 CATGTGTAAA ATTGACGCAT GTGTTTTATC GGTCTGTATA TCGAGGTTTA TTTATTAATT
     2821 TGAATAGATA TTAAGTTTTA TTATATTTAC ACTTACATAC TAATAATAAA TTCAACAAAC
     2881 AATTTATTTA TGTTTATTTA TTTATTAAAA AAAACAAAAA CTCAAAATTT CTTCTATAAA
     2941 GTAACAAAAC TTTTATGAGG GACAGCCCCC CCCCAAAGCC CCCAGGGATG TAATTACGTC
     3001 CCTCCCCCGC TAGGGGGCAG CAGCGAGCCG CCCGGGGCTC CGCTCCGGTC CGGCGCTCCC
     3061 CCCGCATCCC CGAGCCGGCA GCGTGCGGGG ACAGCCCGGG CACGGGGAAG GTGGCACGGG
     3121 ATCGCTTTCC TCTGAACGCT TCTCGCTGCT CTTTGAGCCT GCAGACACCT GGGGGGATAC
     3181 GGGGAAAAGG CCTCCACGGC CAGCTCTAGA CCTGCAGGCA CTAGAGGGTA TATAATGGAA
     3241 GCTCGACTTC CAGCTTGGCA ATCCGGTACT GTACCGGTca cttgagacga ttaaATGGAC
     3301 AGCAtgcgtc tcaAGTCCgc caccatggac cactacctcg acattcgctt gcgaccggac
     3361 ccggaatttc ccccggcgca actcatgagc gtgctcttcg gcaagctcca ccaggccctg
     3421 gtggcacagg gcggggacag gatcggcgtg agcttccccg acctcgacga aagccgctcc
     3481 cggctgggcg agcgcctgcg cattcatgcc tcggcggacg accttcgtgc cctgctcgcc
     3541 cggccctggc tggaagggtt gcgggaccat ctgcaattcg gagaaccggc agtcgtgcct
     3601 caccccacac cgtaccgtca ggtcagtcgg gttcaggcga aaagcaatcc ggaacgcctg
     3661 cggcggcggc tcatgcgccg gcacgatctg agtgaggagg aggctcggaa acgcattccc
     3721 gatacggtcg cgagagcctt ggacctgccc ttcgtcacgc tacgcagcca gagcaccgga
     3781 cagcacttcc gtctgttcat ccgccacggg ccgttgcagg tgacggcaga ggaaggagga
     3841 ttcacctgtt acgggttgag caaaggaggt ttcgttccct ggttcggatc ccccaagaag
     3901 aagaggaaag tctgatcagt agggttgtaa aggtttttct tttcctgaga aaacaacctt
     3961 ttgttttctc aggttttgct ttttggcctt tccctagctt taaaaaaaaa aaagcaaaac
     4021 tcaccgaggc agttcgtatc ggtctgcgaa cactgccgta taggcagcGT CGAGGGACCT
     4081 AATAACTTCG AACTAAACTT GTTTATTGCA GCTTATAATG GTTACAAATA AAGCAATAGC
     4141 ATCACAAATT TCACAAATAA AGCATTTTTT TCACTGCATT CTAGTTGTGG TTTGTCCAAA
     4201 CTCATCAATG TATCTTATCA TGTCTGGAAT TGACTCAAAT GATGTCAATT AGTCTATCAG
     4261 AAGCTCATCT GGTCTCCCTT CCGGGGGACA AGACATCCCT GTTTAATATT TAAACAGCAG
     4321 TGTTCCCAAA CTGGGTTCTT ATATCCCTTG CTCTGGTCAA CCAGGTTGCA GGGTTTCCTG
     4381 TCCTCACAGG AACGAAGTCC CTAAAGAAAC AGTGGCAGCC AGGTTTAGCC CCGGAATTGA
     4441 CTGGATTCCT TTTTTAGGGC CCATTGGTAT GGCTTTTTCC CCGTATCCCC CCAGGTGTCT
     4501 GCAGGCTCAA AGAGCAGCGA GAAGCGTTCA GAGGAAAGCG ATCCCGTGCC ACCTTCCCCG
     4561 TGCCCGGGCT GTCCCCGCAC GCTGCCGGCT CGGGGATGCG GGGGGAGCGC CGGACCGGAG
     4621 CGGAGCCCCG GGCGGCTCGC TGCTGCCCCC TAGCGGGGGA GGGACGTAAT TACATCCCTG
     4681 GGGGCTTTGG GGGGGGGCTG TCCCTGATAT CTATAACAAG AAAATATATA TATAATAAGT
     4741 TATCACGTAA GTAGAACATG AAATAACAAT ATAATTATCG TATGAGTTAA ATCTTAAAAG
     4801 TCACGTAAAA GATAATCATG CGTCATTTTG ACTCACGCGG TCGTTATAGT TCAAAATCAG
     4861 TGACACTTAC CGCATTGACA AGCACGCCTC ACGGGAGCTC CAAGCGGCGA CTGAGATGTC
     4921 CTAAATGCAC AGCGACGGAT TCGCGCTATT TAGAAAGAGA GAGCAATATT TCAAGAATGC
     4981 ATGCGTCAAT TTTACGCAGA CTATCTTTCT AGGGTTAATC TAGCTGCATC AGGATCATAT
     5041 CGTCGGGTCT TTTTTCCGGC TCAGTCATCG CCCAAGCTGG CGCTATCTGG GCATCGGGGA
     5101 GGAAGAAGCC CGTGCCTTTT CCCGCGAGGT TGAAGCGGCA TGGAAAGAGT TTGCCGAGGA
     5161 TGACTGCTGC TGCATTGACG TTGAGCGAAA ACGCACGTTT ACCATGATGA TTCGGGAAGG
     5221 TGTGGCCATG CACGCCTTTA ACGGTGAACT GTTCGTTCAG GCCACCTGGG ATACCAGTTC
     5281 GTCGCGGCTT TTCCGGACAC AGTTCCGGAT GGTCAGCCCG AAGCGCATCA GCAACCCGAA
     5341 CAATACCGGC GACAGCCGGA ACTGCCGTGC CGGTGTGCAG ATTAATGACA GCGGTGCGGC
     5401 GCTGGGATAT TACGTCAGCG AGGACGGGTA TCCTGGCTGG ATGCCGCAGA AATGGACATG
     5461 GATACCCCGT GAGTTACCCG GCGGGCGCGC TTGGCGTAAT CATGGTCATA GCTGTTTCCT
     5521 GTGTGAAATT GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG CATAAAGTGT
     5581 AAAGCCTGGG GTGCCTAATG AGTGAGCTAA CTCACATTAA TTGCGTTGCG CTCACTGCCC
     5641 GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT G
//