LOCUS       sequence-393031-        6654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized V. cholerae TniQ from a CMV 
            promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6654)
  AUTHORS   Lampe GD, King RT, Halpin-Healy TS, Klompe SE, Hogan MI, Vo PLH, 
            Tang S, Chavez A, Sternberg SH
  TITLE     Targeted DNA integration in human cells without double-strand breaks
            using CRISPR-associated transposases.
  JOURNAL   Nat Biotechnol. 2023 Mar 29. doi: 10.1038/s41587-023-01748-1.
  PUBMED    36991112
REFERENCE   2  (bases 1 to 6654)
  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: "Nat 
            Biotechnol. 2023 Mar 29. doi: 10.1038/s41587-023-01748-1."
COMMENT     Sequence Label: pSL2620 (pTniQ_Vch)
FEATURES             Location/Qualifiers
     source          1..6654
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(207..225)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(208..225)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(245..262)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    251..475
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      521..949
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(608..627)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     818..839
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(958..978)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        963..1292
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1143..1278
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1205..1224
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1359..2153
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1413..1432)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2023..2042
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2327..2448
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2364..2383)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2418..2437
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2497..2513)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2497..2513)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2510..2532)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2521..2537
                     /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)."
     promoter        complement(join(2545..2551,2552..2569,2570..2575))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2590..2611
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2728..2745)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2899..3487)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2979..2998)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3658..4449,4450..4518))
                     /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     4281..4300
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4519..4623)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4698..4717)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4889..5268
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5269..5472
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5423..5443
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5517..5536
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5517..5535
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5567..5576
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5612..5632
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 gatcgcctct atggtggagc agcgcatcct gaagcccaat agaaagtcca agcctaactc
       61 tccactggac gtgaccgatt acctgttcca ctttggcgac atcttctgcc tgtggctggc
      121 cgagtttcag tccgatgagt tcaaccggag cttctacgtg agccggtggt aattacattg
      181 gaagtggata atctagaggg ccctattcta tagtgtcacc taaatgctag agctcgctga
      241 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
      301 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
      361 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
      421 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
      481 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
      541 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
      601 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
      661 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
      721 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
      781 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
      841 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
      901 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
      961 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     1021 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     1081 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     1141 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     1201 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     1261 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     1321 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     1381 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     1441 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     1501 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     1561 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     1621 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     1681 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     1741 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     1801 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     1861 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     1921 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     1981 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     2041 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     2101 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     2161 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     2221 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     2281 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     2341 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     2401 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     2461 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     2521 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     2581 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     2641 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     2701 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     2761 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     2821 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     2881 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2941 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3001 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3061 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3121 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3181 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3241 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3301 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     3361 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3421 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3481 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3541 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3601 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     3661 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3721 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     3781 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     3841 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3901 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3961 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4021 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4081 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4141 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4201 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4261 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4321 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4381 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4441 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4501 gaaatgttga atactcatac tcttcctttt tcattattat tgaagcattt atcagggtta
     4561 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     4621 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     4681 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     4741 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     4801 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     4861 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
     4921 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     4981 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     5041 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     5101 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     5161 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     5221 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     5281 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     5341 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     5401 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     5461 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
     5521 acgactcact atagggagac ccaagcttgg taccgagctc ggatccgcca ccatgggcaa
     5581 acggacagcc gacggaagcg agttcgagtc accgaaaaag aagcgaaaag ttggctccgg
     5641 aatgttcctc cagaggccaa agccctacag cgacgagtcc ctggagtctt tctttatcag
     5701 agtggccaat aagaacggct atggcgatgt gcacaggttc ctggaggcca ccaagcggtt
     5761 cctccaggac atcgatcaca acggctacca gacctttcca acagacatca ccaggatcaa
     5821 tccctatagc gccaagaaca gctcctctgc ccgcacagcc tccttcctga agctggccca
     5881 gctgaccttt aatgagccac ctgagctgct gggactggcc atcaatcgga caaacatgaa
     5941 gtactcccct tctaccagcg ccgtggtgag aggagcagag gtgttcccac ggtccctgct
     6001 gagaacccac agcatcccat gctgtcccct gtgcctgaga gagaacggct acgccagcta
     6061 tctgtggcac ttccagggct acgagtattg tcactcccac aatgtgcctc tgatcaccac
     6121 atgctcttgt ggcaaggagt ttgactacag ggtgagcggc ctgaagggca tctgctgtaa
     6181 gtgcaaggag ccaatcaccc tgacaagccg cgagaatggc cacgaggccg cctgtaccgt
     6241 gtccaactgg ctggccggcc acgagtctaa gcctctgcca aacctgccca agtcctatag
     6301 atggggactg gtgcactggt ggatgggcat caaggactcc gagttcgatc acttctcttt
     6361 tgtgcagttc tttagcaact ggcctcggag cttccactcc atcatcgagg acgaggtgga
     6421 gtttaatctg gagcacgccg tggtgtccac ctctgagctg cggctgaagg atctgctggg
     6481 cagactgttc tttggcagca tcaggctgcc agagcgcaat ctccagcaca acatcatcct
     6541 gggcgagctg ctgtgctacc tggagaaccg cctgtggcag gacaagggcc tgatcgccaa
     6601 tctgaagatg aacgccctgg aggccacagt gatgctgaat tgttccctgg atca
//
