LOCUS       pKG03471.pIVT-Bx        3972 bp    DNA     circular SYN 10-JAN-2025
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3972)
  AUTHORS   ISU Biology IT
  TITLE     Direct Submission
  JOURNAL   Exported Jan 10, 2025 from SnapGene 8.0.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3972
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    164..783
                     /label=pBR322_Origin
                     /note="color: #00ffff"
     rep_origin      180..768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     575..590
                     /label=oTA073 Xho_addition_rev
                     /note="color: black; sequence: 
                     catcgagcatatgctcgagaaagcgccacgct; added: 2021-01-11"
     primer_bind     complement(645..660)
                     /label=oTA072 Xho_addition_for
                     /note="color: black; sequence: 
                     gcacatagtcgacctccctcgtgcgctct; added: 2021-01-11"
     primer_bind     669..688
                     /label=oSEQ067 pBR322-ori_fwd
                     /note="color: black; sequence: GGGAAACGCCTGGTATCTTT; added:
                     2024-07-28"
     primer_bind     complement(786..824)
                     /label=DP-409
                     /note="color: black; sequence: 
                     CCTTATAGTGAGTCGTATTAGGCCAGGAACCGTAAAAAG; added: 2024-05-19"
     primer_bind     805..842
                     /label=eGFP-IVT-F
                     /note="color: black; sequence: 
                     AGCTATAATACGACTCACTATAGGGctcctgggcaacgtgctg; added: 
                     2022-10-28"
     primer_bind     805..842
                     /label=DP-404
                     /note="color: black; sequence: 
                     TAATACGACTCACTATAAGGctcctgggcaacgtgctg; added: 2024-05-19"
     promoter        805..821
                     /label=T7 promoter
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    822..823
                     /label=CleanCap AG
                     /note="color: #a6acb3"
     5'UTR           825..886
                     /label=beta-globin 5' UTR
                     /note="color: #a6acb3"
     primer_bind     complement(870..886)
                     /label=pHA-b-globin-IVT-R
                     /note="color: black; sequence: gaccggtagcgtgcttt; added: 
                     2023-06-06"
     primer_bind     887..907
                     /label=Bxb1-pPV2-FWD
                     /note="color: black; sequence: 
                     GCCAGCAACGGGTCTCAaatgcgacccgccaccatgcc; added: 2024-06-14"
     CDS             903..2459
                     /codon_start=1
                     /label=bxb1
                     /note="color: #993366"
                     /translation="MPKKKRKVYPYDVPDYAGSRALVVIRLSRVTDATTSPERQLESCQ
                     QLCAQRGWDVVGVAEDLDVSGAVDPFDRKRRPNLARWLAFEEQPFDAIVAYRVDRLTRS
                     IRHLQQLVHWAEDHKKLVVSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAA
                     HFNIRAGKYRGSLPPWGYLPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHD
                     LNRRGVLSPKDYFAQLQGREPQGRKWSATALKRSMISEAMLGYATLNGKTVRDDDGAPL
                     VRAEPILTREQLEALRAELVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHP
                     RYRCRSMGFPKHCGNGTVAMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAIELAEVN
                     AELVDLTSLIGSPAYRAGSPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFG
                     DWWREQDTAAKNTWLRSMNVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGM
                     S"
     CDS             906..926
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     CDS             join(927..952,953)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This feature has 2 segments:
                        1: 927 .. 952 / #cc99b2
                        2: 953 .. 953 / #cc99b2"
                     /translation="YPYDVPDYA"
     CDS             join(954..1175,1176..1178,1179..1639,1640..1642,1643..2078,
                     2079..2081,2082..2459)
                     /codon_start=1
                     /label=Bxb1
                     /note="This feature has 7 segments:
                        1:  954 .. 1175 / #993366
                        2: 1176 .. 1178 / #ffcc99 / V74A-evo
                        3: 1179 .. 1639 / #993366
                        4: 1640 .. 1642 / #ffcc99 / E229K-ee
                        5: 1643 .. 2078 / #993366
                        6: 2079 .. 2081 / #ffcc99 / V375I-ee
                        7: 2082 .. 2459 / #993366"
                     /translation="GSRALVVIRLSRVTDATTSPERQLESCQQLCAQRGWDVVGVAEDL
                     DVSGAVDPFDRKRRPNLARWLAFEEQPFDAIVAYRVDRLTRSIRHLQQLVHWAEDHKKL
                     VVSATEAHFDTTTPFAAVVIALMGTVAQMELEAIKERNRSAAHFNIRAGKYRGSLPPWG
                     YLPTRVDGEWRLVPDPVQRERILEVYHRVVDNHEPLHLVAHDLNRRGVLSPKDYFAQLQ
                     GREPQGRKWSATALKRSMISEAMLGYATLNGKTVRDDDGAPLVRAEPILTREQLEALRA
                     ELVKTSRAKPAVSTPSLLLRVLFCAVCGEPAYKFAGGGRKHPRYRCRSMGFPKHCGNGT
                     VAMAEWDAFCEEQVLDLLGDAERLEKVWVAGSDSAIELAEVNAELVDLTSLIGSPAYRA
                     GSPQREALDARIAALAARQEELEGLEARPSGWEWRETGQRFGDWWREQDTAAKNTWLRS
                     MNVRLTFDVRGGLTRTIDFGDLQEYEQHLRLGSVVERLHTGMS"
     primer_bind     complement(1160..1187)
                     /label=Bxb1-V74A-REV
                     /note="color: black; sequence: 
                     ggcgacgatggcatcaaagggctgttcc; added: 2024-06-23"
     primer_bind     complement(1160..1187)
                     /label=Bxb1-V74D-REV
                     /note="color: black; sequence: 
                     ggcgacgatgtcatcaaagggctgttcc; added: 2024-06-14"
     primer_bind     1167..1197
                     /label=Bxb1-V74A-FWD
                     /note="color: black; sequence: 
                     ccctttgatgccatcgtcgcctacagagtgg; added: 2024-06-23"
     primer_bind     1167..1196
                     /label=Bxb1-V74D-FWD
                     /note="color: black; sequence: 
                     ccctttgatgacatcgtcgcctacagagtg; added: 2024-06-14"
     misc_feature    1176
                     /label=evo-PASSIGE-V74A
                     /note="color: #c0c0c0; direction: RIGHT"
     primer_bind     complement(1196..1224)
                     /label=Bxb1-I87L-REV
                     /note="color: black; sequence: 
                     gatgtcgcagtgagcgggtcagccggtcc; added: 2024-06-14"
     primer_bind     1205..1233
                     /label=Bxb1-I87L-FWD copy
                     /note="color: black; sequence: 
                     gacccgctcactgcgacatctccagcagc; added: 2024-06-14"
     misc_feature    1215..1217
                     /label=bioarchiv-I87L
                     /note="doi: https://doi.org/10.1101/2024.06.10.598370"
                     /note="color: #ffff00; direction: RIGHT"
     primer_bind     complement(1624..1651)
                     /label=Bxb1-E229K-REV
                     /note="color: black; sequence: 
                     gcactccattttcttccctgtggctctc; added: 2024-06-23"
     primer_bind     1632..1661
                     /label=Bxb1-E229K-FWD
                     /note="color: black; sequence: 
                     cagggaagaaaatggagtgctactgcactg; added: 2024-06-23"
     misc_recomb     1642..1643
                     /label=eePASSIGE-E229K
                     /note="color: #c0c0c0; direction: RIGHT"
     primer_bind     complement(2062..2091)
                     /label=Bxb1-V375I-REV
                     /note="color: black; sequence: 
                     ctgccagctcgatagcggagtcagatcctg; added: 2024-06-23"
     primer_bind     2070..2100
                     /label=Bxb1-V375I-FWD
                     /note="color: black; sequence: 
                     gactccgctatcgagctggcagaagtcaatg; added: 2024-06-23"
     misc_feature    2256..2258
                     /label=bioarchiv-E434G
                     /note="color: #ffff00; direction: RIGHT"
     primer_bind     complement(2437..2456)
                     /label=CAG_Bxb1_GG_Rev
                     /note="color: black; sequence: 
                     tgcagaagacaacttcggacatcccagtgtgcagtc; added: 2024-03-06"
     primer_bind     complement(2441..2463)
                     /label=Bxb1-pPV2-REV
                     /note="color: black; sequence: 
                     CTGACGTCTAGGTCTCTgttgctaggacatcccagtgtgc; added: 
                     2024-06-14"
     primer_bind     2464..2487
                     /label=pHA-b-globin-IVT-F
                     /note="color: black; sequence: ctcgagctcaagcttcgaattcac; 
                     added: 2023-06-06"
     primer_bind     2464..2483
                     /label=3-UTR-F
                     /note="color: black; sequence: ctcgagctcaagcttcgaat; added:
                     2023-02-09"
     3'UTR           2561..2955
                     /label=beta-globin-3'UTR
                     /note="human beta-globin polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(2659..2694)
                     /label=IVT-mRNA-R-from-paper
                     /note="color: black; sequence: 
                     ttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt
                     tttttttttttttttttttttttttttttttttttttttttttttttttttttttttgc
                     aatgaaaataaatgttttttattaggcagaat; added: 2022-10-28"
     primer_bind     complement(2928..2970)
                     /label=DP-405
                     /note="color: black; sequence: 
                     caaaatccgattcggtaaaatacagcatagcaaaactttaacc; added: 
                     2024-05-19"
     primer_bind     complement(2928..2956)
                     /label=3-UTR-R
                     /note="color: black; sequence: 
                     ccgcgtaaaatacagcatagcaaaactttaacc; added: 2023-02-09"
     primer_bind     2941..2983
                     /label=DP-406
                     /note="color: black; sequence: 
                     ctatgctgtattttaccgaatcggattttgtttttaatatttc; added: 
                     2024-05-19"
     misc_feature    2956..2984
                     /label=3' UTR
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     complement(2963..2992)
                     /label=DP-370
                     /note="color: black; sequence: 
                     tttttttttgaaatattaaaaacaaaatcc; added: 2024-05-03"
     primer_bind     complement(2984..3045)
                     /label=IVT-mRNA-R-from-paper
                     /note="color: black; sequence: 
                     ttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt
                     tttttttttttttttttttttttttttttttttttttttttttttttttttttttttgc
                     aatgaaaataaatgttttttattaggcagaat; added: 2022-10-28"
     misc_feature    2985..3106
                     /label=polyA-2x60_G
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     complement(3047..3106)
                     /label=IVT-mRNA-R-from-paper
                     /note="color: black; sequence: 
                     ttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt
                     tttttttttttttttttttttttttttttttttttttttttttttttttttttttttgc
                     aatgaaaataaatgttttttattaggcagaat; added: 2022-10-28"
     misc_feature    3107..3116
                     /label=linearize plasmid w/ MluI for IVT
                     /note="color: #a6acb3"
     primer_bind     complement(3139..3164)
                     /label=DP-407
                     /note="color: black; sequence: TTTTGAGACACgtgccacctggccct; 
                     added: 2024-05-19"
     primer_bind     3143..3181
                     /label=DP-408
                     /note="color: black; sequence: 
                     ccaggtggcacGTGTCTCAAAATCTCTGATGTTACATTG; added: 2024-05-19"
     primer_bind     3144..3170
                     /label=oMJ020
                     /note="color: black; sequence: 
                     tgatatctCTTAGACTGCAGGTGGCCCGTGTCTCAAAATCTCTG; added: 
                     2022-01-26"
     primer_bind     3153..3173
                     /label=oMJ001
                     /note="color: black; sequence: 
                     CGCTCGAAGACTGCAGGTGCCCGTGTCTCAAAATCTCTGATG; added: 
                     2022-01-26"
     primer_bind     3153..3173
                     /label=oMJ005
                     /note="color: black; sequence: 
                     CGCTCGCTGACTGCAGGTGCCCGTGTCTCAAAATCTCTGATG; added: 
                     2022-01-26"
     primer_bind     3153..3173
                     /label=oMJ009
                     /note="color: black; sequence: 
                     TACTCGAAGACTGCAGGTGCCCGTGTCTCAAAATCTCTGATG; added: 
                     2022-01-26"
     primer_bind     3153..3173
                     /label=oMJ013
                     /note="color: black; sequence: 
                     TACTcgctGACTGCAGGTGCCCGTGTCTCAAAATCTCTGATG; added: 
                     2022-01-26"
     misc_feature    3154..3177
                     /label=pENTR2B.Seq.REV
                     /note="color: #ff00ff"
     primer_bind     complement(3166..3187)
                     /label=oTA069 mIR-backbone_junction_rev
                     /note="color: black; sequence: ctTGTGCAATGTAACATCAGAG; 
                     added: 2021-01-11"
     primer_bind     complement(3166..3187)
                     /label=oSEQ044 pENTR_rev_seq
                     /note="color: black; sequence: cttgtgcaatgtaacatcagag; 
                     added: 2024-07-28"
     primer_bind     complement(3239..3259)
                     /label=oSEQ005 pENTR_rev
                     /note="25nm	STD"
                     /note="color: black; sequence: ggctcataacaccccttgtat; 
                     added: 2021-01-11"
     CDS             join(3253..3972,1..90)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /note="color: #ccffcc"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3278..3299)
                     /label=oSEQ023 attl2_rev
                     /note="color: black; sequence: TTGGAATTTAATCGCGGCCTCG; 
                     added: 2021-01-11"
     primer_bind     complement(3286..3309)
                     /label=Kan_rev_SEQ
                     /note="color: black; sequence: AGCATCCATGTTGGAATTTAATCG; 
                     added: 2021-07-14"
     primer_bind     complement(3676..3705)
                     /label=oTA099 GG_entry_upstream_fwd
                     /note="color: black; sequence: 
                     ttgcgcctgagctagACGAAATACGCGATC; added: 2021-01-07"
     primer_bind     3676..3705
                     /label=oTA102 GG_entry_downstream_rev
                     /note="color: black; sequence: 
                     GATCGCGTATTTCGTctagctcaggcgcaa; added: 2021-01-07"
ORIGIN
        1 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
       61 tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacattat
      121 tcagattggg ccccgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
      181 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
      241 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
      301 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
      361 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
      421 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
      481 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
      541 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      601 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
      661 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
      721 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
      781 gcggcctttt tacggttcct ggcctaatac gactcactat aaggctcctg ggcaacgtgc
      841 tggtctgtgt gctggcccat cactttggca aagcacgcta ccggtcaatg cgacccgcca
      901 ccatgccaaa aaagaaaaga aaagtgtatc cctatgatgt ccccgattat gccggttcaa
      961 gagccctggt cgtgattaga ctgagccgag tgacagacgc caccacaagt cccgagagac
     1021 agctggaatc atgccagcag ctctgtgctc agcggggttg ggatgtggtc ggcgtggcag
     1081 aggatctgga cgtgagcggg gccgtcgatc cattcgacag aaagaggagg cccaacctgg
     1141 caagatggct cgctttcgag gaacagccct ttgatgccat cgtcgcctac agagtggacc
     1201 ggctgacccg ctcaattcga catctccagc agctggtgca ttgggctgag gaccacaaga
     1261 aactggtggt cagcgcaaca gaagcccact tcgatactac cacacctttt gccgctgtgg
     1321 tcatcgcact gatgggcact gtggcccaga tggagctcga agctatcaag gagcgaaaca
     1381 ggagcgcagc ccatttcaat attagggccg gtaaatacag aggctccctg cccccttggg
     1441 gatatctccc taccagggtg gatggggagt ggagactggt gccagacccc gtccagagag
     1501 agcggattct ggaagtgtac cacagagtgg tcgataacca cgaaccactc catctggtgg
     1561 cacacgacct gaatagacgc ggcgtgctct ctccaaagga ttattttgct cagctgcagg
     1621 gaagagagcc acagggaaga aaatggagtg ctactgcact gaagagatct atgatcagtg
     1681 aggctatgct gggttacgca acactcaatg gcaaaactgt ccgggacgat gacggagccc
     1741 ctctggtgag ggctgagcct attctcacca gagagcagct cgaagctctg cgggcagaac
     1801 tggtcaagac tagtcgcgcc aaacctgccg tgagcacccc aagcctgctc ctgagggtgc
     1861 tgttctgcgc cgtctgtgga gagccagcat acaagtttgc cggcggaggg cgcaaacatc
     1921 cccgctatcg atgcaggagc atggggttcc ctaagcactg tggaaacggg acagtggcca
     1981 tggctgagtg ggacgccttt tgcgaggaac aggtgctgga tctcctgggt gacgctgagc
     2041 ggctggaaaa agtgtgggtg gcaggatctg actccgctat cgagctggca gaagtcaatg
     2101 ccgagctcgt ggatctgact tccctcatcg gatctcctgc atatagagct gggtccccac
     2161 agagagaagc tctggacgca cgaattgctg cactcgctgc tagacaggag gaactggagg
     2221 gcctggaggc caggccctct ggatgggagt ggcgagaaac cggacagagg tttggggatt
     2281 ggtggaggga gcaggacacc gcagccaaga acacatggct gagatccatg aatgtccggc
     2341 tcacattcga cgtgcgcggt ggcctgactc gaaccatcga ttttggcgac ctgcaggagt
     2401 atgaacagca cctgagactg gggtccgtgg tcgaaagact gcacactggg atgtcctagc
     2461 aacctcgagc tcaagcttcg aattcacccc accagtgcag gctgcctatc agaaagtggt
     2521 ggctggtgtg gctaatgccc tggcccacaa gtatcactaa gctcgctttc ttgctgtcca
     2581 atttctatta aaggttcctt tgttccctaa gtccaactac taaactgggg gatattatga
     2641 agggccttga gcatctggat tctgcctaat aaaaaacatt tattttcatt gcaatgatgt
     2701 atttaaatta tttctgaata ttttactaaa aagggaatgt gggaggtcag tgcatttaaa
     2761 acataaagaa atgaagagct agttcaaacc ttgggaaaat acactatatc ttaaactcca
     2821 tgaaagaagg tgaggctgca aacagctaat gcacattggc aacagcccct gatgcctatg
     2881 ccttattcat ccctcagaaa aggattcaag tagaggcttg atttggaggt taaagttttg
     2941 ctatgctgta ttttaccgaa tcggattttg tttttaatat ttcaaaaaaa aaaaaaaaaa
     3001 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaagaaaa aaaaaaaaaa
     3061 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaacgcg tcgaggggaa
     3121 ttaattcttg aagacgaaag ggccaggtgg cacgtgtctc aaaatctctg atgttacatt
     3181 gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat
     3241 acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac
     3301 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     3361 acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     3421 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     3481 atgcctcttc cgaccatcaa gcattttatc cgtactcctg gtgatgcatg gttactcacc
     3541 actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     3601 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     3661 tgtcctttta acagcgatcg cgtatttcgt ctagctcagg cgcaatcacg aatgaataac
     3721 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     3781 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     3841 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     3901 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     3961 ttttctcctt ca
//
