LOCUS       sequence-434908-        6266 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of SUMO tagged human gamma S crystallin with Q16C 
            mutation to act as a biological control for ligation experiment.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6266)
  TITLE     Completion of previous SUMO tagged human crystallin submission plus 
            plasmids for native chemical ligation to produced gamma S crystallin
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6266)
  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            "Unpublished"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: PE-SUMO CRYGS Q16C
FEATURES             Location/Qualifiers
     source          1..6266
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     77..96
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        77..95
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    96..120
                     /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)."
     RBS             135..157
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             170..187
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             200..487
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated and modified)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /note="modified to prevent cleavage by eukaryotic SUMO 
                     proteases"
                     /translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR
                     LMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
     CDS             1074..1091
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1144..1162)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1158..1205
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1242..1697
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1329..1348)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1539..1560
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1723..1827
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1828..1896,1897..2688)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2046..2065)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2859..3447
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3348..3367
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3601..3618
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3633..3772
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3698..3717)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3858..3880
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3874..4065)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4874..5956)
                     /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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5918..5937
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5957..6034)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6240..6259
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat
       61 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
      121 cctctagaaa taattttgtt taactttaag aaggagatat accatgggtc atcaccatca
      181 tcatcacggg tccctgcagg actcagaagt caatcaagaa gctaagccag aggtcaagcc
      241 agaagtcaag cctgagactc acatcaattt aaaggtgtcc gatggatctt cagagatctt
      301 cttcaagatc aaaaagacca ctcctttaag aaggctgatg gaagcgttcg ctaaaagaca
      361 gggtaaggaa atggactcct taagattctt gtacgacggt attagaattc aagctgatca
      421 ggcccctgaa gatttggaca tggaggataa cgatattatt gaggctcacc gcgaacagat
      481 tggaggttct aaaactggaa ccaagattac tttctatgaa gacaaaaatt tttgtggccg
      541 tcgctatgac tgtgattgcg actgtgcaga tttccacaca tacctaagtc gctgcaactc
      601 cattaaagtg gaaggaggca cctgggctgt ttatgaaagg cccaactttg ctgggtacat
      661 gtacatctta ccacagggag agtaccctga ataccagcgt tggatgggcc tcaacgaccg
      721 cctcagctcc tgcagagctg ttcatctgcc tagtggaggc cagtataaga ttcagatctt
      781 tgagaaaggg gattttagtg gtcagatgta tgaaaccacc gaagattgcc cttccatcat
      841 ggagcaattt cacatgcgag agatccactc ctgtaaggtg ctggagggtg tctggatttt
      901 ctatgagcta cccaactacc gtggcaggca gtacctcctg gacaagaagg agtaccggaa
      961 gcccatcgat tggggtgcag cctccccagc tgtccagtct ttccgccgca ttgtggagta
     1021 atctagagga tccgaattcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc
     1081 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     1141 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     1201 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     1261 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     1321 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     1381 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1441 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1501 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1561 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     1621 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     1681 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1741 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1801 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1861 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     1921 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     1981 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     2041 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     2101 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     2161 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     2221 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     2281 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     2341 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     2401 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     2461 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     2521 aaatctggag ccggtgagcg tggttctcgc ggtatcattg cagcactggg gccagatggt
     2581 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2641 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2701 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     2761 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2821 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2881 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2941 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3001 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3061 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3121 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3181 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3241 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3301 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3361 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3421 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3481 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3541 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3601 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     3661 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     3721 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     3781 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     3841 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     3901 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     3961 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     4021 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     4081 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     4141 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     4201 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     4261 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     4321 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     4381 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     4441 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     4501 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     4561 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     4621 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     4681 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     4741 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     4801 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     4861 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4921 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     4981 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     5041 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     5101 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     5161 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     5221 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     5281 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     5341 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     5401 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     5461 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     5521 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     5581 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     5641 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     5701 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     5761 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     5821 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     5881 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     5941 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     6001 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     6061 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     6121 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     6181 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     6241 gtgatgtcgg cgatataggc gccagc
//
