LOCUS       Exported                6000 bp ds-DNA     circular SYN 24-NOV-2025
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    NEBuilder.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6000)
  TITLE     NEBuilder-generated Construct
  JOURNAL   Exported 20-JAN-2020 from NEBuilder https://nebuilder.neb.com
REFERENCE   2  (bases 1 to 6000)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Nov 25, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     NEBuilder-generated oligos (UPPERCASE = gene-specific, lowercase = 
            overlap) Ser_sfGFP(5')_fwd: 
            aactttaagaaggagatataccatgtctATGAGCAAAGGCGAAGAATTATTTAC 
            Ser_sfGFP(5')_fwd 3'Tm: 63.2  3'Ta: 64.1
            Ser_sfGFP(5')_rev: tcgacagtttTGACTGCGTACTCAGGTAG
            Ser_sfGFP(5')_rev 3'Tm: 63.1  3'Ta: 64.1
            sfGFP(A206K)_3p_fwd: tacgcagtcaaaaCTGTCGAAAGACCCGAATG 
            sfGFP(A206K)_3p_fwd 3'Tm: 63.0  3'Ta: 60.9
            sfGFP(A206K)_3p_rev: agtggtggtggtggtggtgctcgagTTAAACATGATGGTGGTGATG 
            sfGFP(A206K)_3p_rev 3'Tm: 59.9  3'Ta: 60.9.
FEATURES             Location/Qualifiers
     source          1..6000
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      1..589
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC
                     origin of replication; high-copy-number 
                     ColE1/pMB1/pBR322/pUC origin of  replication"
                     /note="/Transferred_From=old MFM16,
                     pET28b-His6-TEV-sfGFP-ASCC3_DxxD (pMFM004), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy, 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003), 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V, 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp, 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002), 
                     pET28b_RH_ISG15_GyrA, pET29b-IGIP1490, pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V, pMFM003 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG, to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/X-Transferred_From=pET28b-His6-TEV-sfGFP-ASCC3_DxxD
                     (pMFM004); pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005); 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy; 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003); 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V; 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp; 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002); 
                     pET28b_RH_ISG15_GyrA; pET29b-IGIP1490; pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V; to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/X-Transferred_Similarity=100.00%"
                     /note="color: #ffff00"
     misc_feature    complement(775..914)
                     /label=bom
                     /note="basis of mobility region from pBR322"
                     /note="/Transferred_From=pET29b-IGIP1490"
                     /note="/Transferred_Similarity=100.00%"
                     /note="color: #a6acb3; direction: LEFT"
     misc_feature    824^825
                     /label=TAT
                     /note="Geneious type: Editing History Deletion"
                     /note="/Original_Bases=TAT"
                     /note="color: #a6acb3"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop; rop"
                     /product="Rop protein, which maintains
                     plasmids at low copy number; Rop protein, which maintains 
                     plasmids at low copy number"
                     /label=rop
                     /note="/Transferred_From=pET28b-sfGFP-SGG-LPETGG_His6V 
                     (pMFM002)
                     translation, old MFM16, pET28b-His6-TEV-sfGFP-ASCC3_DxxD 
                     (pMFM004), pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy, 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003), 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V, 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp, 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002), 
                     pET28b_RH_ISG15_GyrA, pET29b-IGIP1490, pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V, pMFM003 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG, to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/Transferred_Translation=MTKQEKTALNMARFIRSQTLTLLEKLN
                     ELDAD EQADICESLHDHADELYRSCLARFGDDGENL; 
                     VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHADELYRSCLARFGD 
                     DGENL*"
                     /note="/Transferred_Translation_Similarity=100.00%"
                     /note="/X-Transferred_From=pET28b-His6-TEV-sfGFP-ASCC3_DxxD
                     (pMFM004); pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005); 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy; 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003); 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V; 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp; 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002); 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002) translation; 
                     pET28b_RH_ISG15_GyrA; pET29b-IGIP1490; pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V; to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/X-Transferred_Similarity=100.00%"
                     /note="/X-Transferred_Translation=MTKQEKTALNMARFIRSQTLTLLEK
                     LNELD ADEQADICESLHDHADELYRSCLARFGDDGENL; 
                     VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHADELYRSCLARFGD 
                     DGENL*"
                     /note="/X-Transferred_Translation_Similarity=100.00%"
                     /note="color: #993366"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /note="/Transferred_From=old MFM16,
                     pET28b-His6-TEV-sfGFP-ASCC3_DxxD (pMFM004), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy, 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003), 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V, 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp, 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002), 
                     pET28b_RH_ISG15_GyrA, pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V, pMFM003 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG, to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/Transferred_Translation=VKPVTLYDVAEYAGVSYQTVSRVVNQA
                     SHVSA 
                     KTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQL 
                     GASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFL 
                     DVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLT 
                     RNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRV 
                     GADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVS 
                     LVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*"
                     /note="/X-Transferred_From=pET28b-His6-TEV-sfGFP-ASCC3_DxxD
                     (pMFM004); pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005); 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy; 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003); 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V; 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp; 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002); 
                     pET28b_RH_ISG15_GyrA; pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V; to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/X-Transferred_Similarity=100.00%"
                     /note="/X-Transferred_Translation=VKPVTLYDVAEYAGVSYQTVSRVVN
                     QASHV 
                     SAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRAD 
                     QLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPAL 
                     FLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKY 
                     LTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGL 
                     RVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLP 
                     VSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ*"
                     /note="color: #993366"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(3099..3176)
                     /gene="lacI; lacI"
                     /label=lacI promoter
                     /note="/Transferred_From=old MFM16,
                     pET28b-His6-TEV-sfGFP-ASCC3_DxxD (pMFM004), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy, 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003), 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V, 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp, 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002), 
                     pET28b_RH_ISG15_GyrA, pET29b-IGIP1490, pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V, pMFM003 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG, to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/X-Transferred_From=pET28b-His6-TEV-sfGFP-ASCC3_DxxD
                     (pMFM004); pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005); 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy; 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003); 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V; 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp; 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002); 
                     pET28b_RH_ISG15_GyrA; pET29b-IGIP1490; pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V; to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/X-Transferred_Similarity=100.00%"
                     /note="color: #ffffff; direction: LEFT"
     promoter        3485..3503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase; 
                     promoter for bacteriophage T7 RNA polymerase"
                     /note="/Transferred_From=old MFM16,
                     pET28b-His6-TEV-sfGFP-ASCC3_DxxD (pMFM004), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy, 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003), 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V, 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp, 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002), 
                     pET28b_RH_ISG15_GyrA, pET29b-IGIP1490, pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V, pMFM003 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG, to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/X-Transferred_From=pET28b-His6-TEV-sfGFP-ASCC3_DxxD
                     (pMFM004); pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005); 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy; 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003); 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V; 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp; 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002); 
                     pET28b_RH_ISG15_GyrA; pET29b-IGIP1490; pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V; to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/X-Transferred_Similarity=100.00%"
                     /note="color: #ffffff"
     protein_bind    complement(3504..3528)
                     /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)."
                     /note="/Transferred_From=pET29b-IGIP1490"
                     /note="/Transferred_Similarity=100.00%"
                     /note="color: #31849b; direction: LEFT"
     RBS             3543..3565
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
                     /note="/Transferred_From=old MFM16,
                     pET28b-His6-TEV-sfGFP-ASCC3_DxxD (pMFM004), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy, 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003), 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V, 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp, 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002), 
                     pET28b_RH_ISG15_GyrA, pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V, pMFM003 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG, to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/X-Transferred_From=pET28b-His6-TEV-sfGFP-ASCC3_DxxD
                     (pMFM004); pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005); 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy; 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003); 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V; 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp; 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002); 
                     pET28b_RH_ISG15_GyrA; pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V; to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/X-Transferred_Similarity=100.00%"
                     /note="color: #a6acb3"
     CDS             3572..4342
                     /label=MS-sfGFP(A206K)-SGG-SRT-His6
                     /note="/created_by=matthiasmuhar"
                     /note="/modified_by=matthiasmuhar"
                     /note="color: #993366; direction: RIGHT"
     misc_feature    3581..4291
                     /label=sfGFP_core
                     /note="/created_by=matthiasmuhar"
                     /note="color: #a6acb3"
     CDS             4097..4099
                     /label=G174
                     /note="/created_by=matthiasmuhar"
                     /note="color: #993366; direction: RIGHT"
     misc_feature    4301..4318
                     /label=Srt recognition site
                     /note="/Transferred_From=pET28b-sfGFP-SGG-LPETGG_His6V 
                     (pMFM002)
                     - ORF translation, pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002) 
                     translation, pET28b-sfGFP-SGG-LPETGG_His6V Copy - ORF 
                     translation, old MFM16, pET28b-sfGFP-SGG-LPETGG_His6V 
                     (pMFM002), pMFM002 pET28b-sfGFP-SGG-LPETGG_His6V"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/Transferred_Translation_Similarity=100.00%"
                     /note="/X-Transferred_From=pET28b-sfGFP-SGG-LPETGG_His6V
                     (pMFM002); pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002) - ORF 
                     translation; pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002) 
                     translation; pET28b-sfGFP-SGG-LPETGG_His6V Copy - ORF 
                     translation; pMFM002 pET28b-sfGFP-SGG-LPETGG_His6V"
                     /note="/X-Transferred_Similarity=100.00%"
                     /note="/X-Transferred_Translation_Similarity=100.00%"
                     /note="/created_by=matthiasmuhar"
                     /note="color: #a6acb3"
     CDS             4319..4336
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="/Transferred_From=pET28b-His6-TEV-sfGFP-ASCC3_DxxD
                     (pMFM004) translation, pET28b-His6-TEV-sfGFP-ASCC3_RxxG 
                     (pMFM003) - ORF translation, 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V - ORF 
                     translation, pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002) - ORF 
                     translation, pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002) 
                     translation, pET28b-sfGFP-SGG-LPETGG_His6V Copy - ORF 
                     translation, to clone pET28b-His6-TEV-sfGFP-ASCC3_RxxG - 
                     ORF translation, 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V, 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002), pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/Transferred_Translation=HHHHHH"
                     /note="/Transferred_Translation_Similarity=100.00%"
                     /note="color: #993366"
                     /translation="HHHHHH"
     terminator      4433..4480
                     /label=T7 terminator
                     /note="transcription terminator for
                     bacteriophage T7 RNA polymerase"
                     /note="/Transferred_From=pET28b-His6-TEV-sfGFP-ASCC3_DxxD
                     (pMFM004), pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy, 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003), 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V, 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp, 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002), 
                     pET28b_RH_ISG15_GyrA, pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V, pMFM003 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG, to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/Transferred_Similarity=100.00%"
                     /note="color: #ffffff"
     rep_origin      4517..4972
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication;
                     arrow  indicates direction of (+) strand synthesis; arrow 
                     indicates direction of (+) strand synthesis; f1 
                     bacteriophage origin of replication"
                     /note="/Transferred_From=old MFM16,
                     pET28b-His6-TEV-sfGFP-ASCC3_DxxD (pMFM004), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy, 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003), 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V, 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp, 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002), pET29b-IGIP1490, 
                     pMFM002 pET28b-sfGFP-SGG-LPETGG_His6V, pMFM003 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG, to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/X-Transferred_From=pET28b-His6-TEV-sfGFP-ASCC3_DxxD
                     (pMFM004); pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005); 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy; 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003); 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V; 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp; 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002); pET29b-IGIP1490; 
                     pMFM002 pET28b-sfGFP-SGG-LPETGG_His6V; to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/X-Transferred_Similarity=100.00%"
                     /note="color: #ffff00"
     misc_feature    4963
                     /label=T
                     /note="Geneious type: Editing History Replacement"
                     /note="/Original_Bases=T"
                     /note="color: #a6acb3"
     misc_feature    4972^4973
                     /label=C
                     /note="Geneious type: Editing History Deletion"
                     /note="/Original_Bases=C"
                     /note="color: #a6acb3"
     CDS             complement(5064..5879)
                     /codon_start=1
                     /gene="aph(3')-Ia;
                     aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase; aminoglycoside
                     phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in
                     bacteria or G418 (Geneticin(R)) in eukaryotes; confers 
                     resistance to kanamycin in bacteria or G418 (Geneticin(R)) 
                     in eukaryotes"
                     /note="/Transferred_From=old MFM16,
                     pET28b-His6-TEV-sfGFP-ASCC3_DxxD (pMFM004), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005), 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy, 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003), 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V, 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp, 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002), 
                     pET28b_RH_ISG15_GyrA, pET29b-IGIP1490, pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V, pMFM003 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG, to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/Transferred_Similarity=100.00%"
                     /note="/Transferred_Translation=MSHIQRETSCSRPRLNSNMDADLYGYK
                     WARDN 
                     VGQSGATIYRLYGKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRT 
                     PDDAWLLTTAIPGKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFR 
                     LAQAQSRMNNGLVDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIF 
                     DEGKLIGCIDVGRVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFH 
                     LMLDEFF; 
                     MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKPDAPELFLKHGK 
                     GSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTAFQVLEEYPDS 
                     GENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASDFDDERNGWP 
                     VEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIADRYQDLAIL 
                     WNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
                     /note="/X-Transferred_From=pET28b-His6-TEV-sfGFP-ASCC3_DxxD
                     (pMFM004); pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005); 
                     pET28b-His6-TEV-sfGFP-ASCC3_KxxK (pMFM005) Copy; 
                     pET28b-His6-TEV-sfGFP-ASCC3_RxxG (pMFM003); 
                     pET28b-His6-TEV-sfGFP-LoopC-SGG-LPETGG_His6V; 
                     pET28b-His6-TEV-sfGFP-pep_V5 temp; 
                     pET28b-sfGFP-SGG-LPETGG_His6V (pMFM002); 
                     pET28b_RH_ISG15_GyrA; pET29b-IGIP1490; pMFM002 
                     pET28b-sfGFP-SGG-LPETGG_His6V; to clone 
                     pET28b-His6-TEV-mCherry"
                     /note="/X-Transferred_Similarity=100.00%"
                     /note="/X-Transferred_Translation=MSHIQRETSCSRPRLNSNMDADLYG
                     YKWAR 
                     DNVGQSGATIYRLYGKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFI 
                     RTPDDAWLLTTAIPGKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRV 
                     FRLAQAQSRMNNGLVDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNL 
                     IFDEGKLIGCIDVGRVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQ 
                     FHLMLDEFF; 
                     MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKPDAPELFLKHGK 
                     GSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTAFQVLEEYPDS 
                     GENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASDFDDERNGWP 
                     VEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIADRYQDLAIL 
                     WNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
                     /note="color: #ccffcc"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     3481 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     3541 attttgttta actttaagaa ggagatatac catgtctatg agcaaaggcg aagaattatt
     3601 tacgggcgta gtgcctattt tagtggaact ggatggcgat gtgaatggac acaagtttag
     3661 cgtgcgtggc gaaggtgaag gcgatgcgac aaatggcaag ctgacgctga aatttatctg
     3721 tacgaccggt aaactgccgg ttccctggcc gaccctggtt accaccctga catacggtgt
     3781 gcaatgcttc gcccgctatc cagatcacat gaaacagcat gactttttca agagcgcgat
     3841 gcccgaaggc tacgttcaag agcgtaccat cagcttcaaa gatgacggca cttataaaac
     3901 ccgcgcggaa gtaaagtttg aaggtgatac actggtcaat cgcatcgaac tgaagggtat
     3961 tgactttaaa gaagatggca acatcttagg ccataaatta gagtataact tcaactcaca
     4021 caatgtgtac attacggcag acaaacagaa gaacggtatt aaggccaact tcaagattcg
     4081 tcataacgta gaagatggta gcgtccagtt agcggatcac tatcaacaga atacccctat
     4141 tggcgatggt ccagttctgc tgccggataa tcactacctg agtacgcagt caaaactgtc
     4201 gaaagacccg aatgagaaac gcgaccatat ggttctgtta gaattcgtaa ctgccgcagg
     4261 cattacccac ggcatggacg agctgtacaa aagcggtggc ctgccggaaa ccggcggtca
     4321 tcatcaccac catcatgttt aactcgagca ccaccaccac caccactgag atccggctgc
     4381 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     4441 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     4501 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4561 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4621 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4681 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     4741 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4801 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     4861 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     4921 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     4981 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5041 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     5101 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     5161 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     5221 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     5281 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     5341 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     5401 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     5461 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     5521 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     5581 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     5641 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     5701 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     5761 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     5821 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     5881 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     5941 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
