LOCUS       sequence-433253-        6320 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of SUMO tagged human beta A4 crystallin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6320)
  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  JOURNAL   Unpublished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 6320)
  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            "Unpublished"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 CRYBA4
FEATURES             Location/Qualifiers
     source          1..6320
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    40..64
                     /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             79..101
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             114..131
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             144..431
                     /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             1072..1089
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1142..1160)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1156..1203
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1240..1695
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1327..1346)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1537..1558
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1721..1825
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1826..1894,1895..2686)
                     /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(2044..2063)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2857..3445
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3346..3365
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3599..3616
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3631..3770
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3696..3715)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3856..3878
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3872..4063)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4872..5954)
                     /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     5916..5935
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5955..6032)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6238..6257
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
       61 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggtcatcacc
      121 atcatcatca cgggtccctg caggactcag aagtcaatca agaagctaag ccagaggtca
      181 agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga tcttcagaga
      241 tcttcttcaa gatcaaaaag accactcctt taagaaggct gatggaagcg ttcgctaaaa
      301 gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga attcaagctg
      361 atcaggcccc tgaagatttg gacatggagg ataacgatat tattgaggct caccgcgaac
      421 agattggagg taccctgcaa tgcacaaagt cagcgggacc ctggaagatg gtggtgtggg
      481 atgaggacgg cttccagggc cggcggcacg agttcacggc cgagtgcccc agcgtgctgg
      541 agcttggctt cgagactgtg cgatctttga aagtgctgag tggagcgtgg gtgggcttcg
      601 agcatgctgg cttccaaggg cagcagtaca ttctggaacg aggcgaatat ccaagctggg
      661 atgcctgggg cggcaacacg gcctaccccg ccgagaggct cacctccttc cggcctgcgg
      721 cctgtgctaa ccaccgtgac tcgaggctga caatcttcga gcaagagaac ttcctgggca
      781 agaaaggaga gctgagcgat gactatcctt ccctccaggc catgggatgg gaaggcaatg
      841 aagtagggtc cttccacgtc cactctgggg cctgggtttg ctcccagttt ccgggctacc
      901 gaggatttca gtatgtgctg gaatgcgatc accattccgg tgactacaaa catttccggg
      961 agtggggctc tcatgccccg accttccagg tgcagagcat ccgcaggatc cagcagtaat
     1021 ctagaggatc cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac
     1081 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     1141 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1201 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     1261 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1321 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1381 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1441 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1501 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1561 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1621 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1681 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1741 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1801 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     1861 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     1921 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     1981 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     2041 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     2101 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2161 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2221 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     2281 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     2341 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     2401 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2461 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2521 atctggagcc ggtgagcgtg gttctcgcgg tatcattgca gcactggggc cagatggtaa
     2581 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2641 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2701 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2761 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2821 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2881 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2941 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3001 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3061 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3121 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3181 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3241 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3301 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3361 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3421 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3481 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3541 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3601 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3661 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3721 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     3781 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3841 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3901 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     3961 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     4021 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     4081 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     4141 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     4201 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     4261 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     4321 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     4381 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     4441 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     4501 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     4561 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     4621 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     4681 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     4741 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     4801 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     4861 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4921 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     4981 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     5041 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     5101 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     5161 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     5221 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     5281 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     5341 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     5401 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     5461 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     5521 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     5581 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     5641 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     5701 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     5761 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     5821 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     5881 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     5941 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     6001 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     6061 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     6121 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     6181 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     6241 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     6301 gcgtccggcg tagaggatcg
//
