LOCUS       sequence-464169-        6377 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of alginate lyase in Escherichia 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6377)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 6377)
  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"
COMMENT     Sequence Label: pAIP4_5Y33
FEATURES             Location/Qualifiers
     source          1..6377
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(144..959)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(144..959)
                     /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"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     867..886
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     951..970
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1081..1669
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1570..1589
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1823..1840
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1855..1994
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1920..1939)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2080..2102
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2096..2287)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3096..4178)
                     /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     4140..4159
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4179..4256)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4462..4481
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4569..4588
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4569..4587
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4588..4612
                     /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             4627..4649
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4672..4701
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5806..5823
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5876..5894)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5890..5937
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5974..6377,1..52)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6061..6080)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6271..6292
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
       61 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
      121 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
      181 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
      241 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
      301 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
      361 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
      421 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
      481 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
      541 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
      601 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
      661 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
      721 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
      781 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
      841 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
      901 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
      961 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     1021 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     1081 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     1141 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     1201 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     1261 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1321 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1381 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     1441 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     1501 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     1561 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     1621 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     1681 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     1741 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     1801 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     1861 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     1921 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     1981 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2041 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     2101 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     2161 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     2221 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     2281 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     2341 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     2401 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     2461 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     2521 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     2581 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     2641 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     2701 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     2761 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     2821 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     2881 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     2941 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     3001 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     3061 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3121 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3181 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     3241 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     3301 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     3361 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     3421 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     3481 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     3541 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     3601 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     3661 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     3721 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     3781 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     3841 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     3901 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     3961 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     4021 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     4081 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     4141 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     4201 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     4261 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     4321 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     4381 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     4441 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     4501 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc
     4561 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     4621 aataattttg tttaacttta agaaggagat atacatatga tggcgaagat tcatcaccat
     4681 catcaccatc accatcacca cggtagcctg caagattcgg aagtcaacca agaagcaaag
     4741 ccggaagtca agccggaagt gaaaccggaa acccatatta acctgaaagt tagtgacggc
     4801 agctctgaaa ttttctttaa gatcaaaaag accacgccgc tgcgtcgcct gatggaagcg
     4861 tttgccaaac gtcagggcaa ggaaatggat agcctgcgtt tcctgtatga cggtattcgc
     4921 atccaggcag atcaagcgcc ggaagacctg gacatggaag acaacgacat cattgaagcc
     4981 caccgtgaac agattggcgg ctctggaagt aaaacagcta agatagattg gtcacactgg
     5041 acggtcactg taccggaaga gaacccggac aagccaggaa agccgtacag cttgggctat
     5101 ccggaaatcc tgaactacgc cgaagataag atcgcgagca agtacatgta tgacgacccg
     5161 aaagataaat ctgttgtgtt ttatgcgttt ccgtctggtg ttaccaccgc aaatacgcat
     5221 tattcccgtt cggagctgcg tgaaaccatg gaaaccggta gcaataaagt taattggacc
     5281 ttcgctaaag gtggcaaaat gagaggtacg tatgccattg acgacattag caaggagccg
     5341 gacggcaagt acagccgtgt gattatcgct cagattcacg gcgttctgac cgatgagcaa
     5401 cgtgatctga ttggccagaa agacaacaac gcgccaccga ttctgaaggt gtactgggac
     5461 aagggcaaga tccgcgtgaa gaccaaggtt ttgaaagatc ttaacgcgcc ttataaagag
     5521 atgctgtcgg agcacgcgtg gggtgacgac gagggtcgca acttcaaaga gaagatcgat
     5581 ttgaacaccc gttttaccct ggaagtcaag gtttccgacg gccgcatgga ggtgatcctc
     5641 aacgataccg aaagcctggt gtacgatgac atccatatga aaaaatgggg tatttttgaa
     5701 aattacttca aagctggtaa ttatttccaa agcaagaccc cgggtacgtt cgcaaaagtg
     5761 aaaatctact ccctgcaggt tactcacggt tcgtaatgac tcgagcacca ccaccaccac
     5821 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     5881 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     5941 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     6001 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     6061 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     6121 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     6181 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     6241 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     6301 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     6361 ggttaaaaaa tgagctg
//
