LOCUS       sequence-93744-e        4654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For replacement of VSG221 with a blasticidin resistence gene in the 
            VSG221 expression site in T.brucei..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4654)
  TITLE     pBSVSG221KOblast
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4654)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pBSVSG221KOblast
FEATURES             Location/Qualifiers
     source          1..4654
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(126..194,195..986)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(344..363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1157..1745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1646..1665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1899..1916
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2033..2054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2069..2074,2075..2092,2093..2099)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2107..2123
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     primer_bind     2112..2134
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2131..2147
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2131..2147
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2168..2186
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             2967..3365
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(4010..4029)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4011..4029)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4036..4053)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4036..4052)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4045..4067)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4193..4648)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4330..4351)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4542..4561
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
       61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     1921 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     1981 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     2041 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     2101 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     2161 agctcgaaat taaccctcac taaagggaac aaaagctgga gctccaccgc ggtggcggcc
     2221 gctctagatg cagcatatgt aactacctct cgtcacaggc agacgcaagc caaacagctg
     2281 atgattgcac cacgtcttgc agaaactgtt aaaagcagtg ccaatttaga aagaataaaa
     2341 ggaaatgaga gaatgtacaa ataaaggaaa taaaaagaaa aacttttatt gcaccttagg
     2401 atagtgtgga aacgtttaac attaatggag cgtacacaca agtgaatttt ctaaactatt
     2461 agatggcgct caggtttttc tgattatcta aaatgaaggc agcggaaagt gtgccaatgc
     2521 attttaaatg acagatttat tttattaaaa atgagaacac ttagaatatt tcatgtcgat
     2581 tcaagctagc agtaatatgc cagtaaatag tttccattta caagcacaat ttcatcctcc
     2641 ttacgtgatt ctgtagcatt tagttcaaga aaaagcaata aaagaattcg gagacgaaga
     2701 gccggttaga ttgcatttgg acaccgctat acatatgtta agacacacaa gcattctata
     2761 cgtaaaagat ctagtatata ggagcaacgc tctgccaaaa cataatggca agacaaacgg
     2821 ccgtgtttgc cgctgatgct acagaaccag cttaatttcc agaagacgaa aatttgcatg
     2881 ttttcccaca atattttaat tactcttgaa gattgtagtt attcctacgc gacaggatcc
     2941 cccgggctgc aggaattcga taattcatgg ccaagccttt gtctcaagaa gaatccaccc
     3001 tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac tacagcgtcg
     3061 ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta tatcatttta
     3121 ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca
     3181 acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc ccctgcggac
     3241 ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg aaggacagtg
     3301 atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat gtgtgggagg
     3361 gctaagaatt atcaagcttc tattaccgaa tggaaacagc agggaaattt gaagttttaa
     3421 cccagaagca caagcccgct gaaagccaac aacaagcagc aggaacagaa ggcagctgca
     3481 acaagaagga ccaaaatgag tgcaaatccc catgcaaatg gcataacgat gcggaaaaca
     3541 aaaagtgcac attggataag gaggaggcaa aaaaggtagc agatgagact gcaaaagatg
     3601 ggaaaactgg aaacacaaac accacaggaa gcagcaattc ttttgtcatt agcaagaccc
     3661 ctctttggct tgcagttttg cttttttaat ttcccccctc aaatttcccc cctcctttta
     3721 aaattttcct tgctacttga aaactttttg atatatttta acaccaaaac cagccgagat
     3781 tttgtgttct gtgttttgta agttgactgt ctgattgtct agaaatattt tctggcaact
     3841 aaaatttttt tcttttttcc tgtttttttt gtaggtaggt aggaatgggg gggggggggt
     3901 agttaggtag gttagttagg ttagttaggg ggttagttag gggggttagg cttaggatta
     3961 ggattagact taggcttagg actcgagggg gggcccggta cccaattcgc cctatagtga
     4021 gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     4081 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     4141 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggaaattgta
     4201 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     4261 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     4321 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     4381 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     4441 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     4501 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     4561 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     4621 gcgcttaatg cgccgctaca gggcgcgtca ggtg
//
